CHANGES IN LEUKOCYTES AND THROMBOCYTES BEFORE,

DURING AND AFTER CERVICAL CANCER TREATMENT .

SEMINAR (BSS 503)

PRESENTED BY:
ABDULMALIK UMMUAYMAN FOLASADE
205658
HEAMATOLOGY AND BLOOD TRANSFUSION SCIENCES

SUBMITTED TO
THE DEPARTMENT OF MEDICAL LABORATORY SCIENCE, FACULTY OF BASIC
MEDICAL SCIENCE, LADOKE AKINTOLA UNIVERSITY OF TECHNOLOGY,
OGBOMOSHO,OYO STATE, NIGERIA.
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF
BACHELOR OF MEDICAL LABORATORY SCIENCE (BMLS) DEGREE.

SUPERVISED BY: MR ASIMIYU S.O.

JANUARY 2025

CERTIFICATION

This is to certify that this seminar work was carried out by Abdulmalik Ummuayman Folasade
Matriculation number 205658 in the Department of Medical Laboratory Science, Faculty of
Basic Medical Sciences, College of Health Sciences Ladoke Akintola University of Technology,
Ogbomosho under the supervisor of Mr S.O ASIMIYU .
______________________________ ________________________

1
MR S.O ASIMIYU

(Supervisor)
Date

______________________________ ________________________

PROF S.A NASSAR

(Head of Department) Date

DEDICATION

I dedicate this seminar to Allah, the Most Gracious, the Most Merciful, whose guidance and
blessings have been my strength and inspiration. I also dedicate this work to my parents, for their
unwavering love, support, and sacrifices, and to my lecturers, for their invaluable guidance,
encouragement, and knowledge. Thank you all, for being my pillars of strength and motivation.

2
 ACKNOWLEDGMENTS

I am deeply grateful to Allah, the Most Gracious, the Most Merciful, for His guidance and
blessings throughout this seminar journey.
My sincere thanks go to my seminar supervisor Mr S.O Asimiyu , for his invaluable support,
insights, and encouragement.
My sincere appreciation also goes to the Head of the Department , Prof. S.A Nassar and other
lecturers for their leadership assistance and the opportunity given to present this seminar.
Special thanks to my mother for her unwavering support, patience, and understanding, your
contributions, belief in me, and constant motivation have been instrumental in the completion of
this work. I am truly thankful for your continuous encouragement and inspiration.
To the funding wallet, and my biggest motivator, my loving father, plethora of gratitude and love
from me. Words cannot describe how much grateful I am. I say, may you live and prosper to eat
the fruits of your labour.

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TABLE OF CONTENTS

TITLE
PAGE………………………………………………………………………………………………………
……………………………………….1
CERTIFICATION.........................................................................................................................................2
DEDICATION...............................................................................................................................................3
ACKNOWLEDGMENTS.............................................................................................................................4
TABLE OF CONTENTS..............................................................................................................................5
ABSTRACT..................................................................................................................................................7
CHAPTER ONE............................................................................................................................................8
1.0 INTRODUCTION.........................................................................................................................8
CHAPTER TWO.........................................................................................................................................15
2.0 HPV SRAINS; PHYLOGENETICS...............................................................................................15
2.1 CLASSIFICATION ........................................................................................................................16
2.2 TECHNIQUES FOR DETECTION OF HPV................................................................................17
2.3 HPV TYPES AND THEIR LOCATIONS IN THE BODY............................................................18
2.4 THE MOST PREVALENT HPV STRAINS .................................................................................18
2.5 HPV E6 AND E7; THE ONCOPLAYERS
20
2.6 THE PRIMER SEQUENCE FOR PCRS
22
CHAPTER THREE.....................................................................................................................................25

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 3.1 HAEMATOPOIESIS , THROMBOPOIESIS, LYMPHOPOIESIS AND THE LEUKOCYTES
25
3.2 WHITE BLOOD CELLS AND THROMBOCYTES DISORDERS ............................................31
3.2.1 LEUKOCYTOSIS ..................................................................................................................31
3.2.2 THROMBOCYTOSIS..............................................................................................................33
3.2.3. LEUKOPAENIA .....................................................................................................................33
3.2 .3 THROMBOCYTOPAENIA ......................................................................................................34
3.3 SPECIFIC TESTS FOR COMMON HAEMATOLOGICAL DISORDERS.
34
3.4 CHANGES IN LEUKOCYTES AND THROMBOCYTES BEFORE DURING AND AFTER
CERVICAL CANCER TREATMENT
40
3.5 PROTECTION AGAINST CANCER
49
CHAPTER FOUR.......................................................................................................................................52
4.1 CONCLUSION................................................................................................................................52
4.2 RECOMMENDATION..................................................................................................................52
REFERENCES........................................................................................................................................54

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 ABSTRACT

Cervial cancer, predominantly caused by high-risk human papillomavirus (HPV) types,

remains a significant public health burden, particularly in low-resource regions where
screening and treatment opportunities are limited. This study provides an in-depth
exploration of the hematological changes in leukocytes and thrombocytes observed before,
during, and after cervical cancer treatment. It underscores the diagnostic and prognostic
importance of these parameters in evaluating disease progression and therapy
effectiveness. Key discussions include HPV phylogenetics, classification, and the molecular
mechanisms of oncogenesis driven by E6 and E7 proteins, which play pivotal roles in
disrupting cell cycle regulation and immune evasion. The hematological processes of
hematopoiesis, thrombopoiesis, and lymphopoiesis are examined in relation to immune
response and hematological disorders associated with cervical cancer.

Advanced diagnostic techniques, including polymerase chain reaction (PCR) and RNA-
based assays, are highlighted for their utility in detecting HPV variants, assessing viral
oncogene expression, and predicting patient outcomes. The study also reviews the impact of
treatment modalities, such as chemoradiation, on hematological profiles, with a focus on
lymphocyte counts as a prognostic marker for tumor recurrence and survival. Insights into
the interplay between HPV infection, host immunity, and hematological parameters
emphasize the need for integrating hematological assessments into cervical cancer
management. By advocating for early detection, targeted interventions, and routine blood
monitoring, this research aims to enhance therapeutic outcomes, improve patient quality of
life, and reduce the global burden of cervical cancer.

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 CHAPTER ONE

1.0 INTRODUCTION
Overview of cervical cancer
Overall HPV is responsible for 5.2% of all cancers. It is well established that HPV infection
is the primary cause of virtually all cervical cancers and indeed deemed a necessary cause
for the disease, without which, cervical cancer does not arise .A landmark study has shown
that HPV DNA can be found in 99.7% of cervical cancer specimens . Worldwide, the
plethora of HPV types causing cervical cancer varies from one country to another,
however, over 70%, in any given country, are caused by only 2 types, HPV16 and HPV 18.
In a pooled analyses of data from 11 case–control studies of women with histologically
confirmed squamous cell cervical cancer conducted in 9 different countries, HPV DNA
could be detected in 90.7% of the cases by one detection method and in 96.6% by another;
the proportion of control women testing positive were respectively, 13.4% and 15.6%. This
study tested either cytology smears or biopsied tissue and found that the most common
types, in order of frequency, were HPV-16, -18, -45,-31, -33, -52, -58 and -35) .
Worldwide, cervical cancer is the second most common cancer in women and the most
common cancer in women in developing countries . In developed countries, cervical cancer
accounts for 1.7% of all cancers while in developing countries this figure is 7% . In 2000,
the World Health Organisation estimated that there were 470,600 newly diagnosed cases of
cervical cancer and this disease was associated with 233,400 deaths annually −80% of these
deaths occur in developing countries . The higher prevalence of cervical cancer in
developing countries may be largely attributable to the limited access women in these
countries have to screening programs combined with high-risk characteristics such as poor
nutrition and high parity . In the United States, prevalence and mortality rates vary
according to ethnicity . There is a 1.5-fold higher incidence of cervical cancer and a 2-fold
higher cervical cancer-related mortality rate in Black compared with Caucasian women .
Human Papilloma Virus
The papillomaviruses are small double-stranded DNA viruses which infect squamous
epithelia (or cells with the potential for squamous maturation), inducing proliferative
lesions, of which the humble skin wart is a typical example. The viruses are absolutely
species-specific, thus HPVs (human papillomaviruses) only infect humans, rabbit
papillomaviruses only infect rabbits, and so forth. They are also exquisitely tissue-tropic,
with a predilection for infection of either cutaneous or internal squamous mucosal surfaces.
Papillomaviruses are not classified by serotype, but by genotype, and, to date, approx. 130
HPV types have been identified by sequencing the gene encoding the major capsid protein
L1 .
The HPV genome can be divided into three domains: a noncoding URR (upstream
regulatory region) of approx. 1 kb, an early region with ORFs (open reading frames) E6,

7
E7, E1, E2,E4 and E5, and a late region encoding two genes, L1, the major capsid protein,
and L2, the minor capsid protein .

Within a species, the individual viruses show a predilection for either cutaneous or mucosal
surfaces, and, within the groups of skin or mucosal viruses, they can be separated into
high- or low-risk types, depending upon their oncogenic potential. This is shown most
clearly in the genital tract in which there is regular or sporadic infection, with approx. 30–
40 types. These can be divided into those predominately associated with benign ano-genital
warts or condylomata (HPV types 6 and 11, and their relatives), and those associated with
ano-genital cancers and the precursor (intra-epithelial neoplasia) lesions particularly of the
cervix (HPV types 16,18, 31, 33, 35 and 45, and minor types). Virtually 100% of cervical
cancers, the second commonest cancer in women worldwide, contain the HPV DNA
sequences from a highrisk oncogenic genital HPV. The most important players are HPV16,
found in 50–70% of cases, and HPV 18 found in 7–20% of cases . Cervical and other ano-
genital cancers are preceded by a spectrum of intra-epithelial abnormalities.
In the cervix, these form a spectrum ranging from low-grade CIN (cervical intra-epithelial
neoplasia) 1 , moderate CIN2 and high-grade CIN3. High-grade CIN3 lesions are the
obligate precursor lesions for cervical cancer and approx. 90% of high-grade CIN contain
high-risk HPV DNA sequences, and, again, HPV16 is the major player . High-risk genital
HPV infection is very common, and the majority of individuals clear their infection with
time, but a proportion of women, approx. 15%, cannot effectively clear the virus, and the
persistence of a high-risk HPV is the major risk factor for the development of ano-genital
malignancies. To persist, HPV must escape the host immune system.

Papillomavirus infectious cycle

The virus replication cycle is the key to understanding the pathogenesis and
immunobiology of these viruses, since the infectious cycle is in itself an immune-evasion
mechanism inhibiting host detection of virus.Thevirus. The knowledge of this process is
limited in several key areas mainly because of the inability to infect cells in tissue culture
with virus and achieve a complete infectious cycle in vitro.

Infection and vegetative viral growth are absolutely dependent upon a complete
programme of keratinocyte differentiation. Viruses infect primitive basal keratinocytes,
probably targeting stem cells, but high-level expression of viral proteins and viral assembly
occur only in the upper layers of the stratum spinosum and granulosum of squamous
epithelia . Viral gene expression is confined to the keratinocyte, and there is no evidence
that viral genes are expressed in any cell other than keratinocytes. There is a spatial and
temporal pattern of HPV gene expression in the infected epithelium . Virus infects a subset
of primitive basal cells, probably stem cells, at low copy number. At some time after
infection there is a round of viral DNA replication which appears to be independent of the
cell cycle and which amplifies the viral copy number to approx. 50–100 copies/cell. The
infected cell is thought then to leave this primitive stemcell-like compartment to enter the
proliferating compartment of the epithelium. There is then a phase of plasmid or episomal
maintenance, viral gene expression is minimal and, in particular, the expression of

8
oncogenes E6 and E7 is under very tight control with E6/E7 transcripts barely detectable.
When the infected keratinocyte enters the differentiating compartment, exiting the cell
cycle, there is a massive up-regulation of viral gene expression, viral DNA replication
occurs, there is amplification of viral copy number to at least 1000 copies/cell, abundant
expression of the early genes E6 and E7 and expression of the late genes from the late
promoter. It is important to recognize that these events occur in cells that are
differentiating and have exited the cell cycle.

The papillomaviruses encode only one DNA replication enzyme, E1, and, apart from this
and the viral E2 protein, replication is totally dependent on the cellular DNA synthetic
machinery. The problem for the virus is that the cellular DNA polymerases and replication
factors are only produced in mitotically active cells. To solve this problem, the viruses
encode proteins which, in the context of the viral life cycle, reactivate cellular DNA
synthesis in non-cycling cells, inhibit apoptosis and delay the differentiation programme of
the infected keratinocyte, creating an environment that is permissive for viral DNA
replication . replication. The precise details by which this is achieved are imperfectly
understood, but the viral genes central to these functions are E6 and E7, and an
unfortunate, but rare, by-product of this role in high-risk HPV replication is the
deregulation of growth control in the infected cell and the development of cancer.
The replication cycle takes a long time: even in the bestcase scenario, the time from
infection to release of virus will take approx. 3 weeks, since this is the time taken for the
keratinocyte to undergo complete differentiation and desquamate. In reality, the period
between infection and the appearance of lesions is highly variable and can vary from weeks
to months. This virus is basically a hitchhiker joining the keratinocyte at the start of its
journey as a primitive basal cell in the epithelium through to its end as a terminally
differentiated squame. This is a replication strategy in which viral DNA replication and
virus assembly occur in a cell already destined for death by natural causes; there is no viral
induced cytolysis or necrosis and therefore no inflammation. Thus, for most of the duration
of the HPV infectious cycle, there is little or no release into the local milieu of pro-
inflammatory cytokines that are important for DC (dendritic cell) activation and
migration, and the central signals to kick start the immune response in squamous epithelia
are absent . absent. There is no bod-borne phase of the HPV life cycle, and only minimal
amounts of replicating virus are exposed to immune defences; in effect, the virus is
practically invisible to the host. This is a viral strategy that results in persistent chronic
infections as the host remains ignorant of the pathogen for long periods.

HPV infections are exclusively intra-epithelial, and, theoretically, HPV attack should be
detected by the professional APC (antigen-presenting cell) of squamous epithelia, the LC
(Langerhans cell). Virus capsid entry is usually an activating signal for DCs, but there is
evidence that LCs are not activated by the uptake of HPV capsids . LCs, when incubated
with L1 VLPs (virus-like particles) of HPV16 do not initiate epitope-specific immune
responses against L1-derived antigens. In contrast, stromal DCs are activated by VLPs and
stimulate HPV-specific T-cells . Studies in TR (Toll-like receptor) 4-deficient mice suggests
that TLR4 contributes to the recognition of HPV16 VLPs by stromal DCs . There are some

9
intriguing data that suggest that TLR activation may be secondary to VLP binding by cell-
surface glycosaminoglycans .
Epidemiology, transmission and pathogenesis
HPV is a virus with worldwide distribution. Viral warts are very common viral infections,
with an estimated incidence of 7 to 10% in the European population and 1% in the U.S.
population. These numbers increase 50 to 100 times in immunocompromised individuals;
for example, in kidney-transplant recipients, reaching more than 90% 15 years after
transplantation. Warts occur at any age and incidence increases during the school age, with
a peak in adolescence and early adulthood.
HPV is transmitted through direct or indirect contact with an individual who has the
lesion. Dysfunctions in the epithelial barrier by trauma, minor injuries or maceration cause
loss of solution of continuity in the skin, thus allowing viral infection. After inoculation, the
incubation period varies from 3weeks to 8 months. Spontaneous regression is observed in
most cases. Cell-mediated immunity (CMI) seems to have a more important role in the host
response to HPV. A higher prevalence of warts and longer and persistent manifestations
are observed in patients with CMI suppression, such as in kidney-transplant recipients,
individuals with HIV (human immunodeficiency virus), and patients with EV.

The life cycle of an HPV is directly linked to the cell differentiation program of the host
cell. Infection begins when the HPV reaches the cells of the basal layer; there is no viral
replication at this location and the virus just keeps its genome by amplification of a low
number of copies. The replicative phase and protein synthesis occur in the suprabasal
differentiated keratinocytes. Progression time and type of lesion correlates with the
quantity of viral particles detected. Younger warts present a higher viral amount when
compared to old warts. Plantar warts have a higher viral load than common warts. The
center of the lesion appears to be the main site of viral concentration.
In benign lesions, replication of the viral genome is extracromossomal. In malignant
lesions, the viral DNA is integrated into the chromosomes of the host cell and there is no
viral replication. There is inactivation of expression of the E2 protein, which acts as a
negative regulator of the expression of the E6 and E7 oncogenes. The last two promote cell
immortalization by inhibiting cellular proteins that regulate the cell cycle (p53 and pRB),
which are critical fortumor suppression.
IMPORTANCE OF STUDYING THE CHANGES IN HAEMATOLOGICAL PARAMETERS
Haematological parameters are those parameters that are related to the blood and blood
forming organs (Bamishaiye et al., 2009; Waugh et al., 2001). Blood act as a pathological
reflector of the status of exposed patient to infections and other conditions (Olafedehan et
al., 2010). As reported by Isaac and colleagues (2013) animals with good blood composition
are likely to show good performance.

Laboratory tests on the blood are vital tools that help detect any deviation from normal in
the animal or human body (Ogunbajo et al., 2013; Abdullahi, 2009). The examination of
blood gives the opportunity to investigate the presence of several metabolites and other
constituents in the body and it plays a vital role in the physiological, nutrition and
pathological status of an organism (Doyle, 2006 and Aderemi, 2004). According to

10
Olafedehan and colleagues (2010) examining blood for their constituents can provide
important information for the diagnosis and prognosis of diseases in patients.

Blood constituents change in relation to the physiological conditions of health (Togun et al.,
2007). These changes are of value in assessing response of animals to various physiological
situations (Khan and Zafar, 2005). According to Afolabi and colleagues (2010), changes in
haematological parameters are often used to determine various status of the body and to
determine stresses due to environmental, nutritional and/or pathological factors.
Haematological components, which consist of red blood cells, white blood cells or
leucocytes, mean corpuscular volume, mean corpuscular haemoglobin and mean
corpuscular haemoglobin concentration are valuable in monitoring feed toxicity especially
with feed constituents that affect the blood as well as the health status of an individual
(Oyawoye and Ogunkunle, 2004). Red blood cells (erythrocytes) serve as a carrier of
haemoglobin, the major functions of the white blood cell and its differentials are to fight
infections, defend the body by phagocytosis against invasion by foreign organisms and to
produce or at least transport and distribute antibodies in immune response, and blood
platelets are implicated in blood clotting. Low platelet concentration suggests that the
process of clotformation (blood clotting) will be prolonged resulting in excessive loss of
blood in the case of injury. Previous report by Peters and colleagues (2011), indicated that
Packed Cell Volume, haemoglobin and mean corpuscular haemoglobin are major indices
for evaluating circulatory erythrocytes, and are significant in the diagnosis of anaemia and
also serve as useful indices of the bone marrow capacity to produce red blood cells as in
mammals (Chineke et al., 2006 and Awodi et al., 2005).

Objectives;
The primary objective of this study is to investigate the dynamic changes in leukocytes and
thrombocytes before, during, and after cervical cancer treatment to understand their
diagnostic, prognostic, and therapeutic significance. Specifically, the study aims to:
1. Analyze the hematological alterations associated with cervical cancer progression and
treatment, focusing on white blood cell and platelet dynamics.
2. Explore the role of high-risk HPV infections, particularly the oncogenic mechanisms of
E6 and E7 proteins, in driving cervical carcinogenesis and immune evasion.
3. Assess the impact of treatment modalities, such as chemotherapy and radiation, on
hematological parameters and their correlation with treatment outcomes, including tumor
recurrence and survival rates.

11
4. Highlight the significance of advanced diagnostic tools, including polymerase chain
reaction (PCR) and RNA-based assays, in detecting HPV variants and predicting disease
progression.
5. Advocate for the integration of routine hematological assessments in cervical cancer
management to optimize patient care, improve therapeutic outcomes, and reduce cervical
cancer burden globally.
This objective seeks to bridge the gap between hematological research and clinical practice,
providing actionable insights for improving the diagnosis, monitoring, and treatment of
cervical cancer.

CHAPTER TWO

2.0. HPV STRAINS; PHYLOGENETICS

Unlike other viral groups, PVs are not mentioned by serotypes. Classification of these
viruses is based on the species of origin and the degree of relationship between viral

12
genomes.They are classified into different types by comparing the nucleotide sequence of
their viral genome.
PVs are grouped into different genera, which in turn are divided into different species
containing one or more genotypes. Each genotype is grouped into subtypes and variants
depending on the similarity of the sequence in the L1 region. To date, about 100 different
types of HPVs have been fully characterized. In addition to all of these HPVs that have
been fully sequenced, there is a large number of additional types whose genetic sequence
has not been obtained through conventional methods yet.
Different genera of HPVs share less than 60% similarity in the nucleotide sequence of the
major capsid of the protein L1 ORF. Different virus species within the same genus share
about 60% to 70% similarity. It is considered a new HPV type when its genome shows
variations greater than 10% in L1, E6 and E7 genes, and when compared with any
previously known type of HPV. Differences between 2 and 10% represent new subtypes
and variations under 2% are variants of types.

HPVs are grouped into the following genera:

Alpha-, Beta-, Gamma-, Mu- and Nu-papillomavirus. The other genera include PVs
isolated in mammals and birds. The phylogenetic grouping sometimes reflects biological
and pathological similarities, but often there are differences. For example, various types
and species of the same genus can display completely different characteristics and still
belong to the same genus.

Alpha-papillomavirus (Supergroup A)
Those HPVs with tropism for genital epithelium are part of this group. However, some
types belonging to this genus cause common warts. This genus includes the types of HPV
that present high risk for cervical cancer such as HPVs 16 and 18, respectively allocated in
species 9 and 7 of this genus, as well as low-risk types of HPV such as HPVs 6 and 11, both
in species 10. At the same time, this same genus includes non-mucosal HPV types, such as
HPV 7 - associated with cutaneous warts in butchers and handlers of meat, poultry and
fish, HPVs found in species (HPVs 2, 27 and 57), and those found in species 2 (HPVs 3 and
10), which cause common warts on the skin.

Beta-papillomavirus (Supergroup B - Subgroup B1)

They are divided into five different species. HPVs 5 and 8, which belong to species 1 of this
genus, are the most commonly identified in the skin of individuals with epidermodysplasia
verruciformis (EV). This genus also involves cutaneous HPVs detected in the skin of the
population in general without skin lesions, demonstrating the ubiquity and high incidence
of asymptomatic infections.

Gamma-papillomavirus (Supergroup B - Subgroup B2)

13
It covers five different species with seven different types that cause skin lesions: HPVs 4,
48, 50, 60, 88, 65, and 95.

Mu-papillomavirus (Supergroup E)
It includes HPVs 1 and 63. HPV 1 is the most studied member of this group and causes
common and palmar warts.

Nu-papillomavirus (Supergroup E)
It has only one species: HPV 41.

2.1. CLASSIFICATION
Historically, HPVs are grouped considering their tissue tropism for certain types of
epithelium and according to the location where they were first isolated. Based on these
characteristics, there arevthree main groups of HPV: cutaneous, mucosal, and associated
with EV. Mucosal HPVs are divided into low and high risk according to their oncogenic
potential. All types of HPV have tropism for cells of the stratified squamous epithelium,
but there are variations in terms of affinity for different anatomical sites. For example,
HPV-1 is a skin type with a high rate of replication in keratinized epithelium of the palmar
and plantar regions. HPV 16 is a mucosal HPV type with preference for genital areas, and
HPV 11, also mucosal, presents replication in laryngeal and genital epithelium.
This classification is not entirely correct, because genital HPV types can be detected in the
skin and the opposite is also possible.

2.2. TECHNIQUES FOR DETECTION OF HPV

HPV does not grow on conventional culture media and serological diagnostic methods have
limited accuracy. The diagnosis of HPV infection is made by histopathology of lesions or
detection of viral DNA in infected cells.Hybridization techniques and polymerase chain
reaction (PCR) are methods used for HPV detection. Among the hybridization techniques
used are the following:
1) Southern blot has high specificity and sensitivity. It allows estimates of the amount of
DNA in the lesion. It has limitations due to the high diversity of types of HPV, since it does
not detect the DNA of unknown viral sequences.
2) Dot blot and reverse blot are laborious techniques that present similar sensitivity and
good accuracy.
3) In situ hybridization uses radiolabeled probes and allows for topographic localization of
viral DNA in cells and tissues. Although the sensitivity of this technique is limited, it is the
best method to assess the distribution of HPV in lesions and it allows for viral localization
by means of other markers.
4) Non-radioactive hybrid capture: this technique is safe, easy to perform and reproduce. It
presents good accuracy for mucosal lesions.
Polymerase chain reaction (PCR) is the most sensitive method. It is also the most widely
used for viral detection and its main application is related to situations where the amount
of DNA available is limited. First, it is necessary to extract the genetic material to be used.
After extracting the DNA, a mixture (premix) containing deoxyribonucleotide

14
triphosphates (dATP, dCPT, dGTP, dTTP), primers (oligonucleotides), the DNA
polymerase enzyme, and a buffer solution is added. This whole mix is sent to the thermal
cycler, which runs in pre-defined temperature cycles, with specific time periods for each
step of the reaction (denaturation, annealing, extension). The PCR result is visualized as a
band of molecular weight specific for the DNA fragment amplified by electrophoresis on
polyacrylamide or agarose gel, using staining with ethidium bromide.
After amplification of viral DNA by PCR, the material needs to be subjected to a technique
that permits identification of the HPV type. The techniques most often used for typing of
the DNA amplified by means of PCR are: Southern blot, dot blot, reverse hybridization,
restriction enzyme digestion, andvsequencing.

Classification of HPV types into cutaneous, mucosal (genital), cutaneous and / or mucosal
and cutaneous associated with epidermodysplasia verruciformis according to the location
of the lesion that it most often causes .causes.

2.3. HPV TYPES AND THEIR LOCATIONS IN THE BODY

Location HPV types
Cutaneous 1, 4, 41, 48, 60, 63, 65, 76, 77, 88, 95
Mucosal 6, 11, 13, 16, 18, 26, 30, 31, 32, 33, 34, 35, 39, 42, 44, 45,
51, 52, 53, 54, 55, 56, 58, 59, 64, 66, 67, 68, 69, 70, 72, 73, 74,
81, 82, 83, 84,86,87,89
Cutaneous and/or mucosal 2, 3, 7, 10, 27, 28, 29, 40, 43, 57, 61, 62, 78, 91, 94, 101, 103
Cutaneous associated 5, 8, 9, 12, 14, 15, 17, 19, 20/46*, 21, 22, 23, 24, 25, 36, 37,
with Epidermodysplasia Verruciformis 38, 47, 49, 50, 80, 75, 92, 93, 96, 107

2.4. THE MOST PREVALENT HPV STRAINS

A. HPV 6 AND 11
Genomically, HPV-6 and HPV-11 are the two most closely related HPV types found
commonly in mucosal epithelia. This might indicate relatively recent speciation events
which could still have a record in the genomic diversity of today’s viral population. As to
their phenotypes, they are found in a variety of anatomical sites including the epithelia of
the outer and the inner genitalia of both males and females and in the mouth and larynx.
Although categorized as ‘‘low-risk’’ HPV types because of their preferential association
with benign anogenital lesions, HPV-6 and HPV-11 are also regularly found in
malignancies (5, 7, 19, 22, 38, 45, 48). The low-risk epidemiological grouping may stem
from the properties of their E6 and E7 proteins, which have lower binding affinities than
those of ‘‘high-risk’’ HPVs for the p53 and retinoblastoma tumor suppressor
proteins .proteins.

HPV-6b, the reference clone, was originally isolated from a condyloma acuminatum , while
HPV-11 was found in a laryngeal papilloma . Both viruses have subsequently been

15
commonly found in both types of lesions, and they are now considered the most frequently
encountered HPV types that cause benign laryngeal and genital neoplasias. In
epidemiological studies, altered restriction patterns have been found more frequently
among variant forms of HPV-6 than among any other genital HPV type. This phenomenon
gave rise to the subtype classification.

B. HPV 16
HPV-16 is one of the most common sexually transmitted mucosal HPV types. It is also the
HPV type most frequently detected in cervical cancer and it is a prototypic “high-risk”
HPV type . Persistent HPV-16 infection is a strong risk factor for development of cervical
cancer. The HPV-16 virion is simple and consists of a histone-covered, approximately 8-kb
double stranded circular genome, with all genes located on the same strand, enclosed in an
icosahedral capsid made of viral L1 and L2 protein . The genome can be divided into an
early region encoding E1, E2, E4-E7 and a late region encoding the two capsid proteins L1
and L2 . One early and one late promoter, and one early and one late polyadenylation
signal are also found on the HPV genome. The viral life cycle can be divided into an early
stage in which transcription from the early promoter generates mRNAs encoding all early
genes that are subsequently polyadenylated at the early polyA signal (pAE) . ).

Initially E6 and E7 proteins drive cell proliferation by binding to p53 and pRb (and other
factors and interfering with their functions . E1 and E2 interact with each other and
replicate the viral DNA genome by binding to viral DNA through E2, and cellular DNA
polymerase through E1 . High levels of E2 apparently shut down the early promoter
concomitantly with activation of the late promoter brought about by terminal cell
differentiation as the infected cell reaches the higher layers of the epithelium.

The late promoter drives expression of mRNAs encoding E1, E2 and E4 that are
polyadenylated at pAE, and L1 and L2 mRNAs that are polyadenylated at pAL . This
results in the production of progeny virus at the top of the epithelium.

C. HPV 18
High-risk human papillomavirus (HPV) types are the etiological agents of cervical cancer.
HPV18 was first described in1984 and is the prototype member of the alpha-7 HPV species.
Based upon its enrichment in cervical cancer compared to the level in cytologically normal
women and its presence in 16% of cervical cancers worldwide , HPV18 is widely accepted
as the second most carcinogenic HPV type after HPV16.

HPV18 is known to be present in a higher proportion of cervical adenocarcinomas (ADC)

(37%) than cervical squamous cell carcinomas (SCC) (12%), an attribute that is shared by
other members of the alpha-7 species . This suggests a phylogenetic trait denoting a
tendency to cause ADC, and previous studies have suggested that the association with ADC
is driven by particular HPV18 variant lineages .lineages.

16
Most HPV18 infections are asymptomatic and are cleared by the immune system. Factors
that favor a small proportion of HPV18 infections to progress to cervical cancer are poorly
understood, but studies have implicated a role for HPV18 genetic variation. Based upon
common phylogenetic patterns of single-nucleotide polymorphisms (SNPs) in the L1 viral
genomic region,HPV18 variants originally were classified as European (E), Asian
Amerindian (AA), or African (AFR) . This classification has been superseded by a whole
viral genome sequencing approach that has defined three major lineages (A, B, and C) and
additional sublineages (A1 to A5 and B1 to B3) (13) that can be largely translated to the
historical nomenclature (A1 and A2 AA, A3 to A5 E, and B/C AFR) . In the whole-genome
approach, differences of 1.0% define variant lineages, and differences of 0.5 to 0.6 define
sublineages.

2.5. HUMAN PAPILLOMAVIRUS E6 AND E7 – THE ONCOPLAYERS

HPV E6 and E7 viral oncoproteins play the pivotal role in driving the cells toward
oncogenesis. In their process of replicating the viral genome, they can induce all the
hallmarks of a cancer cell, i.e., uncontrolled cellular proliferation, angiogenesis, invasion,
metastasis, and unrestricted telomerase activity along with the evasion of apoptosis and
growth suppressors’ activity. Several in vitro and xenograft studies have also shown cancer
cells to senesce or undergo apoptosis in the absence of E6 and E7 activity (Yamato et al.,
2008; Jabbar et al., 2009), thus proving the absolute requirement of E6 and E7 for
persistence of HPV-mediated cancer. Both E6 and E7 are transcribed polycistronically
from a single promoter located at the 3′ end of the upstream regulatory region (URR).
E6/E7 transcription is under the regulation of several transcription factors such as AP1
and SP1, which functions by binding to the URR region.

E7 was the first oncogene to be discovered, among all the HPV oncogenes. It is a relatively
small phosphoprotein of about 100 amino acids, with three conserved regions 1/2/3
(CR1/2/3). A small portion of CR1 and nearly entire CR2 from the amino terminal holds
sequence similarity with adenovirus (Ad) E1A proteins and large T antigen of SV40
(Phelps et al., 1988). The CR2 domain is composed of poorly conserved sequence followed
by the CR3 region. The CR3 region at the carboxyl terminal end is conserved and encodes
a zinc finger domain containing two CXXC motifs separated by 29 amino acid residues
(Barbosa et al., 1990; McIntyre et al., 1993). It is responsible for the zinc-dependent
dimerization and for mediating E7 interaction with cellular proteins responsible for cell
cycle regulation and apoptosis (p21 and pRb; Ohlenschlager et al., 2006).

On the other hand, E6 is relatively larger protein with 150–160 amino acids coding an 18
kDa protein (Zanier et al., 2012). It is arranged into two zinc finger binding domains by
four Cys-X-X-Cys motifs, which have been found to be responsible for the oncogenicity of
the protein (Nomine et al., 2006). The carboxy terminal domain contains a PDZ-binding
motif responsible for interacting with several cellular proteins (Thomas et al., 2002). Out
of all the types of cellular interactions the oncoproteins undergo, the most significant
interaction is the one where E6 can degrade p53 and in case of E7 is the inhibition of pRb
protein.

17
Besides E6 and E7, E5 also plays a vital role in the process of oncogenesis. E5 is an 83
amino acid hydrophobic membraneassociated protein associated with endoplasmic
reticulum. Initially, E5 from BPV was identified as an oncogene; later, HPV16 E5 was also
proved to be oncogenic, which can induce carcinogenic transformation along with E6. It is
known to induce aberrant cellular proliferation through ligand-mediated activation of
EGFR, inhibit apoptosis through degradation of Fas receptors and prevention of formation
of death domain, and help the carcinogenic cells evade immune trap to progress toward
malignancy (reviewed by Venuti et al., 2011)

Detection of E6 and E7 expression

E6 E7 mRNA was detected by RT –PCR. The amplified regions were both in the exons of
E6 (position in gene sequence 421 –540nt) and E7 genes (642 –774 nt). Total RNA was
obtained from cells with Trizol and chloroform. After RNA identification and DNA
digestion, mRNA was reversely transcripted to singlestranded cDNA with oligodT . cDNA
samples were diluted and subjected to PCR amplification with forward and reverse
primers specific to HPV16 E6, E7 and b-actin .

CR analyses of E6 and E7 cDNA fragments were performed under the following

conditions: denaturation was at 941C for 5 min, followed by 24 –26 PCR cycles to ensure
amplification in the linear stage (denaturation at 941C for 30 s, primer annealing at 581C
for 1 min, prime extension at 681C for 2 min). b-Actin cDNA was detected under almost
the same conditions, with three cycles fewer. For every PCR assay, a negative control and a
positive control were used to avoid possible false positives and false negatives. Amplified
products were separated on 2% agarose gels. Intensities of E6 and E7 bands were
quantified using the Image Gauge software and normalised to those of b-actin bands.
Because E6 proteins were unsuccessfully detected by western blotting with several
commercial anti-E6 antibodies (Butz et al,2003), E6 and E7 proteins were only detected by
immunocytochemistry. Cells were seeded on slides in 12-well plates. After siRNA was
treated for 48 h, 4% paraformaldehyde was used to fix cells on the slides and 0.1% Triton
X-100 was used to break the nuclear member. Subsequently, slides were incubated with
monoclonal antibodies to E6 (Calbiochem, Darmstadt, Germany) diluted 1 : 50 and to E7
(Santa Cruz, Santa Cruz, CA, USA) diluted 1 : 75 separately in PBS for 1 h, and then
incubated with Dako Envision peroxidase for 1 h. Thereafter, 3,30 -diaminobenzidine
tetrahydrochloride was added for visualisation and Mayer’s haematoxylin was used to
counterstaining. Positive cells were indicated by the presence of a distinct brown stain in
the nucleus or cytoplasm of cells. Treatment of blank control was performed by replacing
E6 or E7 antibodies with normal rabbit serum.

Treatment of negative control was performed using C33A (a cervical cancer cell without
HPV infection). The immunoreaction of E6 and E7 protein expression was semi-
quantitatively scored in the same manner as that in previous study (Innocenzi et al, ),)

18
2.6.THE. THE PRIMER SEQUENCE FOR PCRS
Primer. Sequence
E6F 50. -TCAAAAGCCACTGTGTCCTG-30
E6R 50. -CGTGTTCTTGATGATCTGCA-30
E7F 50. -ATTAAATGACAGCTCAGAGGA-30
E7R 50. -GCTTTGTACGCACAACCGAAGC-30
b-ActinF 50. -TTCCAGCCTTCCTTCCTGG-30
b-ActinR 50. -TTGCGCTCAGGAGGAGCAAT-30
msp7708F 50. -TTTTGGTTTGTTTTAATTAA-30
msp161R 50. -ACAACTCTATACATAACTATAATA-30
msp115R 50. -ATCCTAAAACATTACAATTCTCTTTTAATA-30
msp53mR 50. -CGATTCAACCGATTTCGATTACGCCCTT-30
msp53nmR 50. -CAATTCAACCAATTTCAATTACACCCTT-30
Pyro-msp11F 50. -TTTATGTATAAAATTAAGGG-30
F14 50. -ATGTATAAAACTAAGGGCGTAA-30
R113 50. -CCTGAAACATTGCAGTTCTC-30
GAPDH-F 50. -TACTAGCGGTTTTACGGGCG-30
GAPDH-R 50. -TCGAACAGGAGGAGCAGAGAGCGA-30
Abbreviations: GAPDH ¼ glyceraldehyde-3-phosphate dehydrogenase; PCR ¼ polymerase
chain reaction.

It is known that the development of a malignant phenotype requires continuous expression

of the E6 and E7 viral oncogenes. E6 and E7 viral oncoproteins bind and modulate cellular
gene products (p53 and pRb) that play a key role in cell cycle control and DNA repair. The
resulting genomic instability is a necessary condition for cell transformation and
immortalization . Increased expression of these transcripts has been described for both
high-grade squamous intraepithelial lesions (HSIL) and clinical samples of cervical
carcinoma . It is logical, therefore, to hypothesize that E6 and E7 RNA transcripts could
predict disease progression . Although the value of RNA testing has yet to be assessed in
large-scale clinical trials, recent studies are encouraging. Since DNA-based assays cannot
distinguish between transients potentially transforming infections, several studies have
suggested that testing for HPV E6 and E7 RNAs could be more specific.
Taken together, these results suggest that RNA-based assays could have a higher
prognostic value than DNA-based tests and that they could play an important role in future
screening programs.

19
.
Sequences of primers (F and R) and TaqMan probes (P) used for real-time PCR, target
locations, and lengths of amplification products
Primer Sequence (5 to 3) Location(a) Product
size(bp)
HPV 16 E6-F GCACCAAAAGAGAACTGCAATGTT. 85–108.
152
HPV 16 E6-R AGTCATATACCTCACGTCGCAGTA. 197–236
HPV 16 E6-P GGACCCACAGGAGCGACCCAGAAAGTTA 112–139
HPV 16 E7-F CAAGTGTGACTCTACGCTTCGG. 738–759.
81
HPV 16. E7-R GTGGCCCATTAACAGGTCTTCCAA. 796–818
HPV 16. E7-P TGCGTACAAAGCACACACGTAGACATTCGT. 763–792
HPV 18. E6-F CTATAGAGGCCAGTGCCATTCG. 503–524.
79
HPV 18. E6-R TTATACTTGTGTTTCTCTGCGTCG. 558–581
HPV 18. E6-P CAACCGAGCACGACAGGAACGACTCCA. 530–556
HPV 18. E7-F TAATCATCAACATTTACCAGCCCG. 721–744.
113
HPV 18. E7-R CGTCTGCTGAGCTTTCTACTACTA. 810–833
HPV 18. E7-P CGAGCCGAACCACAACGTCACACAATGTT. 745–774
HPV 31, E6-F AAGACCGTTGTGTCCAGAAG. 428–447.
106
HPV 31, E6-R GTCTTCTCCAACATGCTATGC. 511–534
HPV 31, E6-P CGTCCTGTCCACCTTCCTCCTAT. 488–511
HPV 31, E7-F TGTGTTAGATTTGCAACCTGAG. 592–613.
78
HPV 31, E7-R ACATCCTCCTCATCTGAGCT. 669–649
HPV 31, E7-P CAACTGACCTCCACTGTTATGAGCAAT 615–641
HPV 33, E6-F TGCACGACTATGTTTCAAGAC 100–120
132
HPV 33, E6-R CTCAGATCGTTGCAAAGGTTT 211–231
HPV 33, E6-P ATTCCACGCACTGTAGTTCAATGTTGT 179–205
HPV 33, E7-F TTGTAACCTGTTGTCACACTTG 733–754
88
HPV 33, E7-R AGTAGTTGCTGTATGGTTCGTA 799–820
HPV 33, E7-P ACTTGCTGTACTGTTGACACATAAACGA 767–794

20
(a); Nucleotide positions based on genome sequences for HPV-16, -18, -31, and -33
(GenBank accession numbers K02718, X05015, J04353, and M12732, respectively).

CHAPTER THREE

3.1. HAEMATOPOIESIS,
THROMBOPOIESIS ,LYMPHOPOIESISTHROMBOPOIESIS, LYMPHOPOIESIS AND
THE LEUKOCYTES
HAEMATOPOIESIS
Haematopoiesis is the process by which all mature blood cells are produced. It must
balance enormous production needs (the average person produces more than 500 billion
blood cells every day) with the need to regulate the number of each blood cell type in the
circulation. In vertebrates, the vast majority of hematopoiesis occurs in the bone marrow
and is derived from a limited number of hematopoietic stem cells that are multipotent and
capable of extensive self-renewal.
Hematopoietic stem cells give rise to different types of blood cells, in lines called myeloid
and lymphoid. Myeloid and lymphoid lineages both are involved in dendritic cell
formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils,
eosinophils, erythrocytes, and megakaryocytes to platelets. Lymphoid cells include T cells,
B cells, natural killer cells, and innate lymphoid cells.
The definition of hematopoietic stem cell has developed since they were first discovered in
1961. The hematopoietic tissue contains cells with long-term and short-term regeneration
capacities and committed multipotent, oligopotent, and unipotent progenitors.
Hematopoietic stem cells constitute 1:10,000 of cells in myeloid tissue.
HSC transplants are used in the treatment of cancers and other immune system disorder
due to their regenerative properties.

21
Figure 3.1; Diagrammatical representation of haematopoiesis

Haematopoiesis is an ideal system for investigating the developmental relationships

between cells of an organ system: haematopoiesis was the first system for which a
tissuespecific stem cell was identified, and all haematopoietic lineages can be reconstituted
from a single bone-marrow-derived cell, the haematopoietic stem cell (HSC). The study of
haematopoiesis began in the 1960s, when Till and mcCulloch showed that bone marrow
cells injected into irradiated mice gave rise to spleen colonies, and when Bradley and
metcalf showed that bone marrow cells dispersed in semi-solid medium formed
heterogeneous colonies. Later, it was realized that haematopoiesis involves interactions
between developing cells and mesenchymederived stromal cells, which led investigators to
develop culture systems that incorporate adherent non-lymphoid cells to support the
generation of myeloid or lymphoid cells.
THROMBOPOESIS
Thrombopoiesis is the formation of thrombocytes (blood platelets) in the bone marrow.
Thrombopoietin is the main regulator of thrombopoiesis. Thrombopoietin affects most
aspects of the production of platelets. This includes self-renewal and expansion of

22
hematopoietic stem cells, stimulating the increase of megakaryocyte progenitor cells, and
supporting these cells so they mature to become platelet-producing cells.The process of
thrombopoiesis is caused by the breakdown of proplatelets (mature megakaryocyte
membrane pseudopodial projections). During the process almost all of the membranes,
organelles, granules, and soluble macromolecules in the cytoplasm are being consumed.
Apoptosis also plays a role in the final stages of thrombopoiesis by letting proplatelet
processes to occur from the cytoskeleton of actin.

Figure 3.2; Diagrammatical representation of thrombopoiesis

LYMPHOPOESIS
In the case of mammals such as humans (Homo sapiens), lymphopoiesis begins with limited
passive provision from the mother. This includes lymphocytes and immunoglobulin G that
cross the placenta and enter the fetus to provide some protection against pathogens, as well
as leukocytes that come from breast milk and enter circulation via the digestive tract. It is
often not effective in preventing infections in the newborn.

23
However, early in gestation, the developing embryo has begun its own lymphopoiesis from
the fetal liver. Lymphopoiesis also arises from the yolk sac. This is in contrast to the adult
where all lymphocytes originate in the bone marrow.
There are four major types of lymphocytes, along with many sub-types. Scientists have
identified hundreds or thousands of lymphocyte cell types, all of which are generated by
normal or abnormal lymphopoiesis, except for certain artificial strains created in
laboratories through the development of existing strains. Although lymphocytes are usually
considered mature, as seen in blood tests, they are certainly not inert. Lymphocytes can
travel around the body wherever there is a need. When such needs arise, new rounds of
downstream lymphopoiesis, such as cell multiplication and differentiation, may occur,
accompanied by intense mitotic and metabolic activity.

Figure 3.3; Diagrammatical representation of lymphopoiesis

THE LEUKOCYTES
All white blood cells are produced and derived from multipotent cells in the bone marrow
known as hematopoietic stem cells.Leukocytes are found throughout the body, including

24
the blood and lymphatic system. All white blood cells have nuclei, which distinguishes them
from the other blood cells, the anucleated red blood cells (RBCs) and platelets. The
different white blood cells are usually classified by cell lineage (myeloid cells or lymphoid
cells). White blood cells are part of the body's immune system. They help the body fight
infection and other diseases. Types of white blood cells are granulocytes (neutrophils,
eosinophils, and basophils), and agranulocytes (monocytes, and lymphocytes (T cells and B
cells)). Myeloid cells (myelocytes) include neutrophils, eosinophils, mast cells, basophils,
and monocytes.Monocytes are further subdivided into dendritic cells and macrophages.
Monocytes, macrophages, and neutrophils are phagocytic. Lymphoid cells (lymphocytes)
include T cells (subdivided into helper T cells, memory T cells, cytotoxic T cells), B cells
(subdivided into plasma cells and memory B cells), and natural killer cells. Historically,
white blood cells were classified by their physical characteristics (granulocytes and
agranulocytes), but this classification system is less frequently used now. Produced in the
bone marrow, white blood cells defend the body against infections and disease. An excess of
white blood cells is usually due to infection or inflammation. Less commonly, a high white
blood cell count could indicate certain blood cancers or bone marrow disorders.

The number of leukocytes in the blood is often an indicator of disease, and thus the white
blood cell count is an important subset of the complete blood count. The normal white cell
count is usually between 4 × 109/L and 1.1 × 1010/L. In the US, this is usually expressed as
4,000 to 11,000 white blood cells per microliter of blood. White blood cells make up
approximately 1% of the total blood volume in a healthy adult, making them substantially
less numerous than the red blood cells at 40% to 45%. However, this 1% of the blood
makes a large difference to health, because immunity depends on it. An increase in the
number of leukocytes over the upper limits is called leukocytosis. It is normal when it is
part of healthy immune responses, which happen frequently. It is occasionally abnormal,
when it is neoplastic or autoimmune in origin. A decrease below the lower limit is called
leukopenia. This indicates a weakened immune system.

25
Figure 3.4; The types of leukocytes

3.2. WHITE BLOOD CELL ANDCELL AND PLATELETS DISORDERS

3.2.1. Leucocytosis
A.Neutrophilia: Neutrophils are commonly increased during pregnancy and in acute
infections, inflammation, alcohol intoxication, corticosteroid therapy and acute blood loss
or red cell destruction. Additional findings on the full blood count can be helpful to identify
the cause of neutrophilia. The combination of anaemia and neutrophilia occurs in chronic
infection or inflammation, and also in malignant conditions; a high Hct with neutrophilia
suggests polycythaemia vera. Neutrophilia with an increased platelet count occurs in
infectious or inflammatory processes or malignant conditions and during marrow
recovery. Neutrophilia with thrombocytopenia is classically seen in sepsis and occasionally
in microangiopathic haemolytic anaemia. Examination of the peripheral blood film also
provides additional clues to confirm or exclude a particular diagnosis. For example,
neutrophilia with the neutrophils showing heavy cytoplasmic granulation (‘toxic’
granulation) is a common finding in severe bacterial infections. In the absence of any
underlying cause, a high neutrophil count with immature myeloid cells suggests chronic
myelogenous leukaemia (CML), and cytogenetic and molecular studies to look for t(9;22)
and the BCR–ABL1 fusion gene are indicated.

B. Lymphocytosis; Lymphocytosis is a feature of certain infections, particularly infections

in children. It may be especially marked in pertussis, infectious mononucleosis,
cytomegalovirus infection, infectious hepatitis, tuberculosis and brucellosis . Elderly
patients with lymphoproliferative disorders, including chronic lymphocytic leukaemia and

26
lymphomas, often present with lymphadenopathy and a lymphocytosis. Morphology and
immunophenotyping of the cells combined with histological examination of a bone marrow
trephine biopsy specimen (and if necessary other tissue biopsy) are used to classify these
disorders and to give an indication of management and prognosis.
If lymph nodes are enlarged, a lymph node biopsy for histology and
immunohistochemistry may be helpful in diagnosis. It is occasionally difficult to
differentiate between a reactive and a neoplastic lymphocytosis. In this situation,
immunophenotyping, to provide evidence of light chain restriction and polymerase chain
reaction for immunoglobulin or T-cell receptor gene rearrangements, may indicate the
presence of a monoclonal population of lymphocytes, thereby supporting a diagnosis of
neoplastic, rather than reactive, lymphoproliferation.

Causes of Lymphocytosis
1. Infections
• Predominantly viral (commonest is infectious mononucleosis)
• Occasionally bacterial (e.g. pertussis and chronic infections like tuberculosis)
• Unusually parasites (e.g. babesiosis)
2. Stress and Postsplenectomy
3. Smoking
4. Hypersensitivity Reactions
5. Autoimmune Disorders
6. Thymoma
7. Clonal disorders
• Monoclonal B cell lymphocytosis
• Lymphoproliferative disorders especially chronic lymphocytic leukaemia and lymphomas

C. Monocytosis;A slight to moderate monocytosis may be associated with some protozoal,

rickettsial and bacterial infections including malaria, typhus and tuberculosis. Monocytosis
associated with neutrophilia is suggestive of chronic myelomonocytic leukaemia. High
levels of monocytes (monocyte count >1×109/l) in an elderly patient suggest chronic
myelomonocytic leukaemia or sometimes, atypical chronic myeloid leukaemia. These
conditions fall into the myelodysplastic/myeloproliferative neoplasm group of disorders, so
the diagnosis is supported by finding splenomegaly, quantitative and qualitative
abnormalities in other cell lines or a clonal cytogenetic abnormality.

D.Eosinophilia; Eosinophilia is typically associated with parasitic infections, skin diseases

and allergic disorders. Eosinophils have a tendency to infiltrate and damage tissues such as
the heart, lungs and gut, so in patients with eosinophilia, assessment of these organs is
necessary. In most cases, the cause of the eosinophilia is indicated by the clinical history,
which should include details of all medications and foreign travel, and by examination of
the stool and urine for parasites, cysts and ova.
E.Basophilia; basophilia as an isolated finding is unusual. However, it is a common feature
of myeloproliferative neoplasms, and basophils may be particularly prominent in CML. In
this condition, an increasing basophil count may be the first indication of accelerated phase

27
disease. Endocrinopathies such as myxoedema and oestrogen abnormalities, infections and
allergic diseases – and rarely, other haematological malignancies – can also cause
basophilia.

3.2.2. Thrombocytosis
Thrombocytosis can be primary or secondary (reactive) to surgery, infectious and
inflammatory conditions, hyposplenism, blood loss and malignancy, and can occur as a
rebound phenomenon following recovery from marrow suppression. Spurious
thrombocytosis can also occur in severe burns and cryoglobulinaemia because the size of
the red cell fragments or cryoglobulin particles is similar to that of platelets. A moderately
increased platelet count (e.g. 450–800×103/l) often does not indicate a primary
haematological disorder. When there is isolated persistent thrombocytosis in a
myeloproliferative neoplasm the diagnosis is essential thrombocythaemia (providing that
the presence of a BCR–ABL1 fusion gene has been excluded).
Thrombotic or haemorrhagic complications can occur with thrombocytosis but often the
diagnosis is made incidentally. Individuals with essential thrombocythaemia have been
noted to have JAK2 V617F (50%), MPL (10%) or CALR mutations, with the JAK2
mutation being associated with an increased risk of thrombosis.
3.2.3. Leucopenia
A.Neutropenia; Once physiological variation, ethnicity and familial or cyclic neutropenia
have been excluded , the nonhaematological causes of isolated neutropenia to be considered
include overwhelming infection, autoimmune disorders such as systemic lupus
erythematosus, irradiation, drugs (particularly anticancer agents) and large granular
lymphocytic leukaemia. Bone marrow examination may assist in determining whether the
problem is the result of peripheral destruction (increased marrow myeloid precursors) or
stem cell failure (lack of marrow myeloid precursors). Typical marrow appearances occur
in drug-induced neutropenia, in which there is a relative paucity of mature neutrophils and
in infant genetic agranulocytosis (Kostmann syndrome) in which there is maturation arrest
at the promyelocytic stage.
B. Reduced numbers of lymphocytes, monocytes, eosinophils and basophils.
Lymphocytes, eosinophils and basophils may all be reduced by physical stress such as
surgery, trauma and infection. Lymphopenia with neutrophilia is a common combination
of haematological abnormalities in severe acute respiratory syndrome and in many other
patients with acute illness or trauma. Lymphopenia, especially affecting the CD4 cells, may
occur in HIV infection and renal failure. Monocytopenia (monocyte count <0.2×109/1) is
typically found in hairy cell leukaemia,which is also associated with pancytopenia, typical
bone marrow histology and lymphocytes with a characteristic cytology and
immunophenotype.

3.2.4. Thrombocytopenia
Thrombocytopenia is a common isolated finding, and it is important to ensure that the
laboratory result reflects a true reduction in platelet count before embarking on further
diagnostic tests. Frequent causes of spurious thrombocytopenia include blood clots in the

28
sample, platelet clumping and platelet satellitism. Platelet clumping, which is seen on the
blood film, can occur in vitro as the result of a temperaturedependent or anticoagulant-
dependent autoantibody or on slides that have been made directly from a finger prick
sample. True thrombocytopenia is most frequently the result of autoantibodies (i.e.
immune thrombocytopenia), HIV infection, anticancer chemotherapy, other drugs (such as
thiazide diuretics), alcohol excess, hypersplenism and MDS. Heparininduced
thrombocytopenia and thrombosis is a particularly important syndrome to recognise .
The first step in the assessment of patients with thrombocytopenia is the examination of a
blood film. The clinical circumstances, together with blood film and bone marrow
examination, usually enable the various causes of thrombocytopenia to be differentiated.
An association with thrombosis, disturbed renal or hepatic function and haemolytic
anaemia should prompt investigations for other diseases, such as thrombotic
thrombocytopenic purpura and, in a pregnant woman, the HELLP (haemolysis, elevated
liver enzymes, low platelet count) syndrome. The presence of thrombocytopenia with
atypical features on the blood film may prompt a bone marrow examination to exclude
conditions such as acute leukaemia, especially in children.

3.3. SPECIFIC TESTS FOR COMMON HAEMATOLOGICAL DISORDERS

Common haematological disorders are outlined in the following sections with suggestions
for investigations that may be helpful in confirming the diagnosis. The lists are indicative
and are not intended to be exhaustive because the protocols and range of tests provided
locally will depend on the availability of expertise and technology. The investigations
discussed are those that are likely to be available within a general haematology
department.

A. Red cell disorders

1.Microcytic hypochromic anaemias
• Measurement of serum ferritin or iron plus either total iron-binding capacity or
transferrin assay, red cell protoporphyrin or soluble transferrin receptors
• Bone marrow aspirate with staining for iron
• Stool examination for occult blood
• Gastrointestinal imaging and endoscopy, with biopsies if appropriate; rarely, blood loss
studies with 51Cr labelled red cells
• Tests for malabsorption
• Serological tests for coeliac disease (e.g. tissue transglutaminase antibodies)
• Serum lead (if lead poisoning is suspected)
If thalassaemia is suspected:
• HPLC or haemoglobin electrophoresis plus haemoglobin A2 and F measurements
• Haemoglobin H preparation
• Family studies
• DNA analysis (when the diagnosis is clinically important).

29
2. Macrocytic anaemias
If macrocytic, megaloblastic erythroid maturation is demonstrated, further investigations
should be undertaken . If the blood film is typical of megaloblastic anaemia, relevant assays
and further investigations can often indicate the diagnosis without the need for a bone
marrow aspirate. Macrocytosis may also be secondary to conditions such as alcohol excess,
liver disease, MDS, hydroxycarbamide administration and hypothyroidism. Reticulocytosis
from any cause can also increase the MCV.

3.Aplastic anaemia
• Cobalamin and folate assays (although bone marrow hypoplasia is rare)
• Viral studies, particularly for EBV, HIV and hepatitis viruses
• Bone marrow aspirate and trephine biopsy including cytogenetic analysis
• Flow cytometry for glycosylphosphatidylinositolanchored proteins to detect a paroxysmal
nocturnal haemoglobinuria (PNH) clone, followed by urine examination for haemosiderin
if positive
• Peripheral blood gene mutation analysis for dyskeratosis congenita if there are relevant
clinical features or lack of response to immunosuppressive therapy.
If Fanconi anaemia is suspected:
• Studies of sensitivity of chromosomes to breakage by DNA cross-linking agents.

4.Haemolytic anaemias
A haemolytic process may be suspected by the presence of a falling Hb, a reticulocytosis
and jaundice with an increase in unconjugated bilirubin level.

B.White cell disorders

The blood film is often of critical importance in the differential diagnosis of white cell
disorders though it may sometimes be normal (e.g. in some patients with lymphoma or
neutrophil functional defects). Changes in white cell numbers or morphology may occur
rapidly in response to local or systemic disorders. In chronic leukaemias, bone marrow
aspiration may add little to the diagnosis, but the pattern of infiltration of neoplastic cells
seen on trephine biopsy can have diagnostic value or prognostic significance (e.g. in
lymphoma and chronic lymphocytic leukaemia).

1.Acute leukaemia
• Full blood count and peripheral blood film
• Bone marrow aspirate and trephine biopsy
• Blood or marrow immunophenotyping for monitoring minimal residual disease
(cytochemical stains can be used if immunophenotyping is not readily available)
• Cytogenetic analysis
• Molecular studies (e.g. fluorescence in situ hybridisation (FISH) analysis) for
identification of acute lymphoblastic leukaemia (ALL) with hyperdiploidy or ETV6–
RUNX1 fusion, detection of BCR–ABL1 fusion in adults with ALL and detection of other
mutations of specific oncogenes (e.g. NPM1, CEBPA and possibly FLT3 in AML).

30
2.Neutropenia
• Cobalamin and folate assays
• Autoantibody screen including rheumatoid factor and investigations for systemic lupus
erythematosus
• Serial neutrophil counts for cyclical neutropenia
• Tests for antineutrophil antibodies
• Bone marrow aspirate and trephine biopsy
• Flow cytometry for PNH (see aplastic anaemia above)
• Consider the need for clonality studies for investigation for an abnormal T-cell
population.

3.Chronic myelogenous leukaemia

• Full blood count and peripheral blood film
• Bone marrow aspirate
• Cytogenetic analysis
• Molecular studies (e.g. real-time quantitative reverse transcriptase or FISH) for BCR–
ABL1 transcripts
• Neutrophil alkaline phosphatase score using cytochemistry (only if cytogenetic and
molecular genetic analysis are not available).

4.Chronic lymphoproliferative disorders and/or lymphadenopathy

Various specimens can be used for investigations including lymph nodes, bone marrow
aspirates, trephine biopsy cores and peripheral blood and other fluids such as
cerebrospinal fluid, ascitic fluid and pleural aspirates.
• Full blood count and peripheral blood film
• Serum protein electrophoresis and immunoglobulin concentrations
• Plasma uric acid, calcium and lactate dehydrogenase (LDH)
• Serological screening for infectious mononucleosis, cytomegalovirus infection, HIV
infection and toxoplasmosis (if infectious cause suspected) and human T-cell lymphotropic
virus, when clinically relevant
• Bone marrow aspirate and trephine biopsy (to demonstrate the presence and distribution
of abnormal lymphocytes) and/or lymph node or other tissue biopsy
• Flow cytometry immunophenotyping or immunohistochemistry of biopsy specimens
• Cytogenetic or molecular genetic analysis including investigation for immunoglobulin
heavy chain or T-cell receptor gene rearrangement if the diagnosis of lymphoma is in doubt
• Imaging (plain radiographs, ultrasonography, computed tomography scan, magnetic
resonance imaging).

5.Myelomatosis (plasma cell myeloma)

• Full blood count and peripheral blood film
• Serum protein electrophoresis, immunofixation and quantification of immunoglobulins
and any paraprotein
• Urine electrophoresis and immunofixation for Bence–Jones protein (early morning urine
sample and, if positive, quantification on 24h collection)

31
• Serum free light chain quantification and ratio
• Serum albumin, tests of renal function, plasma uric acid, calcium, phosphate and alkaline
phosphatase measurements
• β2 microglobulin quantification• Plasma viscosity
• Bone marrow aspirate (with cytogenetic or FISH analysis if results will influence
treatment decisions, and flow cytometry immunophenotyping or DNA analysis if these
analyses are to be used for monitoring minimal residual disease)
• Trephine biops
• Radiologic skeletal survey.

6.Other disorders
• Thrombocytopenia
• Full blood count and blood film
• Reticulocyte count
• Rh blood group
• Direct antiglobulin test
• HIV test
• Hepatitis screen
• Helicobacter pylori test
• Antinuclear antibodies
• Lupus anticoagulant
• Immunoglobulin profile
C. Myeloproliferative neoplasms
• Full blood count and blood film
• Cobalamin (or B12-binding capacity)
• Uric acid assay
• JAK2 and CALR mutation analysis if PV, ET or primary myelofibrosis is suspected
• Arterial oxygen saturation and carboxyhaemoglobin level (selected patients only)
• Abdominal ultrasound examination
• Cytogenetic analysis
• Bone marrow aspirate and trephine biopsy
• Serum erythropoietin assay
• Red cell and plasma volume (selected patients only).
If splenectomy is contemplated:
• Ferrokinetic and red cell survival studies
• Spleen scan and red cell pool measurement.

3.4. CHANGES IN LEUKOCYTES AND THROMBOCYTES BEFORE, DURING AND

AFTER TREATMENT OF CERVICAL CANCER ; CASE STUDIES
A. Changes in Leukocytes

32
 1.Treatment1. Treatment and total lymphocyte counts examination
Information relating to known prognostic factors in HNSCC was obtained from the
medical records of each patient. Baseline values collected prior to initiation of radiation
and chemotherapy included: complete blood count (CBC), total lymphocyte counts,
Human papillomavirus (HPV) status, ECOG performance status, smoking history, alcohol
dependence, T staging, N staging and overall clinical staging, and primary tumor site. In
addition, the dose and duration of radiation, whether the chemotherapy regimen was
cisplatin or carboplatin based, and treatment responses were collected. CBC’s were
routinely performed monthly after radiation and chemotherapy were initiated and these
values were also recorded.
Total lymphocyte counts were collected before beginning chemoradiation and monthly
thereafter for a total of 24 months. Baseline TLCs were classified as normal (≥1000
cells/mm3) or abnormal (<1000 cell/mm3). Following the initiation of antineoplastic
treatment, the National Cancer Institute’s Common Terminology Criteria for Adverse
Events (CTCAE) (version 4.0) was used to classify the severity of treatment related
lymphopenia.
The TLCs at two months after the initiation of radiation and chemotherapy were
dichotomized to CTCAE grade 0–II versus grade III–V for the relevant analyses. For
patients with missing lymphocyte counts at 2 months, the TLCs at 1 month were used
instead. Progression free survival (PFS) was measured from the date of diagnosis to the
date of tumor recurrence or censored at the date of last follow-up. Overall survival time
was measured from the date of diagnosis to the date of death due to any cause. Survival
was censored if the subject was alive at the time of last follow-up

2.Total2. Total lymphocyte counts over time

The median total lymphocyte count (TLC) for all patients prior to chemoradiation was
1660 cells/mm3 (range 890–3090 cells/mm3) and only three patients (5%) had baseline TLC
<1000 cells/mm3 (890, 940, and 970 cells/mm3). Two months after beginning radiation with
cisplatin (n=53) or carboplatin (n=3) the TLC fell by 73% with 61% patients having TLC
<500 cells/mm3 (median 445 cells/mm3, p<0.0001) . The median TLC remained below
1000 cells/mm3 for the entire one year observation period. Nine patients had missing
lymphocyte counts at 2 months and in these patients the TLCs at 1 month were normal.
There was no significant difference in the baseline demographic, staging, laboratory, or adjuvant
treatment data in patients whose TLCs remained above 500 cells/mm3 or fell significantly at 2
months after chemoradiation treatment.

3.Total3. Total lymphocyte counts with HPV status

For purposes of analysis, patients were divided into two groups depending on HPV status.
The median TLC dropped significantly with chemoradiation in both HPV+ and HPV−
patients. HPV+ patients (n=34) had baseline median TLC of 1610 cells/mm3 which fell to
320 cells/mm3 2 months after beginning chemoradiation. A total of 26 patients (76%) had
TLC <500 cells/mm3 at 2 months. HPV− (n=22) patients started with a median TLC at
1770 cells/mm3 which fell to 550 cells/mm3 with 8 patients (36%) having TLC <500

33
cells/mm3 2 months after beginning chemoradiation .

4.Tumor recurrence
Overall 14 of the 56 patients (25%) had recurrence of tumor and 9 patients (16%) died of
their tumor. HPV+ patients had significantly longer overall survival (OS) (p=0.006) and
PFS (p=0.01) than HPV− patients. Five of 34 HPV+ patients had tumor progression and 2
have died while 9/22 HPV− patients had disease progression and 7 died.
HPV− patients who developed grade III–IV treatment-related lymphopenia (TLC <500
cells/mm3) two months after beginning radiation and chemotherapy had a significant
higher hazard rate of disease progression than those whose TLC remained higher.

5.Observed infections
Total of six patients (11%) had a documented infection within the 12-month observation
period after the initiation of chemoradiation. Four of these were HPV−. Five patients had
post-operative MRSA infections and recovered uneventfully with intravenous vancomycin.
One patient had cellulitis which rapidly resolved with oral antibiotics. Five of these six
patients with infection are alive and one died from tumor progression approximately two
years after diagnosis. The three HIV positive patients did not develop infections during
their one year follow-up period.

6.Baseline characteristics of HPV− patients

The median age of the patients was 60 years (range 37–77) and 68% of the patients were
over the age of 55. Sixty-eight percent were male, 45% were Caucasian, and 73% had an
ECOG performance status of zero. Seventy-seven percent had smoked more than 10 pack
years. All three HIV patients were HPV−. Forty-one percent were stage I–III and 59%
were stage IVA or IVB. Fifty-seven percent were T classification 3–4 and 43% were N
classification 2B-3.
Twenty-three percent patients had an oropharyngeal tumor. Sixty-eight percent had
complete or partial responses . The median total lymphocyte count (TLC) for all HPV−
patients prior to chemoradiation was 1770 cells/mm3 (range 940–3090 cells/mm3) with
91% had baseline TLC ≥1000 cells/mm3. Pre-treatment lymphopenia (TLC <1000
cells/mm3) was present in only two patients (9%). These HPV− patients were well
balanced at the baseline demographic, staging, laboratory, or adjuvant treatment data in
whose TLCs remained above 500 cells/mm3 or fell significantly at 2 months after
chemoradiation treatment.

B. Changes in thrombocytes
1.Cancer and thrombocytopaenia
This case demonstrates a young woman presenting with thrombocytopenia, thrombosis,
high levels of D-dimer, and antibodies to PF4 complexes within 10 days after nine-valent
HPV vaccination. A combination of these features has not previously been reported after
this vaccine.
Recent publications have presented patients with a similar symptom constellation of

34
thrombosis combined with low platelets 5–24 days postvaccination with the ChAdOx1
nCoV-19 and Ad26. COV2.S vaccines. Laboratory results in these patients show a
consistent pattern of antibodies to PF4 complexes with unusually high OD values in
ELISA, as well as heparin-independent platelet activation in a variety of functional platelet
activation assays.
The patient exhibited laboratory features indistinguishable from those of the previously
reported VITT cases; high anti-PF4 antibody levels in ELISA and positive functional
platelet activation tests.
Addition of PF4 enhanced platelet activation, whereas FcɣRIIAblocking abolished
activation, demonstrating a FcɣR-dependent mechanism. Noteworthy, the HemosIL HIT
IgG assay was positive.
This assay does not reliably identify antibodies to PF4 complexes in VITT patient sera,
suggesting that the patient's antibodies differ slightly from the majority of previously
described VITT patients, at least in vitro.
The clinical course with increasing platelet counts rapidly following anticoagulant and
immunosuppressive treatment is consistent with reported VITT cases. We propose this to
be the first documented case of VITT occurring after HPV vaccination. Spontaneous HIT
is an autoimmune condition triggered by recent inflammatory stimuli such as surgery or
infections. VITT may represent spontaneous HIT, with the recent vaccine as the triggering
event. However, the mechanism from inflammation to formation of pathological platelet-
activating antibodies leading to thrombosis and thrombocytopenia remains unknown. Why
some individuals are more vulnerable to these vaccine side effects also remains elusive.

The patient had previously received three doses of a bivalent HPV vaccine without any side
effects. A negative anti-PF4 antibody test 4 months before vaccination further implicates
the nine-valent vaccine as an initial triggering event, as opposed to a recall response. The
bivalent and nine-valent vaccine are very similar, but the production cell lines and the
alum adjuvant formulations are different.

These vaccines are based on a virus-like particle, composed of the major L1 capsid protein
for HPV types and an aluminum-containing adjuvant, whereas ChAdOx1 nCoV-19 and
Ad26.COV2.S are adenoviral vector vaccines. Thus, theories centered on the SARSCoV2
spike protein and adenoviral proteins as factors contributing to antigenic PF4 complex
formation, might need reassessment. Concerns regarding an increase in thromboembolic
events following HPV vaccination have been raised, but follow-up studies adjusting for
preexisting VTE risk factors found no increased risk. Because VITT was just recently
recognized, cases of antiPF4-driven thromboembolism following HPV vaccination might
not have been identified.

However, the patientthe patient is not directly comparable to the usual demographic
receiving this vaccine (i.e., she was slightly older and previously HPV vaccinated). In
addition, she had an active HPV infection, thus the use of a fourth dose was off-label.
Whether her age, the previous vaccine, and/or the underlying infection contributed to the
VITT development are unknown. VITT has only been linked to recombinant adenoviral

35
vector coronavirus disease 2019 vaccines, no other risk factors have been identified and the
age-adjusted incidence is not yet determined.

Mass vaccination campaigns allow for uncovering rare complications. The current
attention to VITT made a timely diagnosis and appropriate treatment possible for the
patient, who showed clear and rapid clinical improvement. That VITT potentially is a
previously underdiagnosed condition, not restricted to recombinant adenoviral vector or
coronavirus disease 2019 vaccines, is conceivable. It is suggested that physicians be aware
of signs of thrombocytopenia and thrombosis after any vaccination, and that this be
monitored in upcoming register vaccine studies.

2.Human papilloma virus vaccine induced thrombocytopenia accompanied by a wide

spectrum of reversible inflammatory responses – a case report
Recently reported a 25-year-old woman who developed thrombocytopenia, venous
thrombosis, elevated D-dimer levels and high levels of platelet-activating antibodies to
platelet factor 4-polyanion complexes 10 days after Gardasil 9 vaccination for human
papillomavirus (HPV). This vaccine is a non-infectious recombinant vaccine prepared from
the purified virus-like particles of the major capsid (L1) protein of HPV types 6, 11, 16, 18,
31, 33, 45, 52 and 58. The patient exhibited clinical and laboratory features in line with the
recently defined VITT syndrome described after adenoviral vector vaccination to prevent
COVID-19 .
To characterize the inflammatory responses in this patient, herein ,presentherein, present
data on inflammatory and related markers four months prior, during and 12 months after
the acute event. We selected markers that will cover a broad spectrum of inflammatory
pathways such as vascular inflammation (i.e. pentraxin (PTX)3 and YKL-40), neutrophil
activation (i.e. neutrophil gelatinase-associated lipocalin (NGAL), S100A8/A9) including
neutrophil extracellular traps (NETs) formation (citrullinated histone 3 (H3Cit)),
inflammation that involves extracellular matrix remodeling (i.e. growth differentiation
factor (GDF)-15 and S100A8/A9) and macrophage activation (i.e. soluble [s]CD163 and
S100A8/A9), and some of these have also been involved in development of VITT .
It was found that there was increased plasma levels of Ykl-40, GDF-15 and PTX3 during
the acute phase in HPV vaccinated patients as compared to the controls (un-complicated
ChAdOx1 nCoV-19 vaccinated (n = 5–8) and not vaccinated (n = 8–11)), but the level did
not reach the levels in the VITT patients (n = 4–5). The macrophage activation marker
sCD163, however, was as high as observed in the VITT patients potentially suggesting
increased M2 tissue macrophage activation. The neutrophil markers NGAL and
S100A8/A9 were higher than the controls pointing toward neutrophil activation, although
not reaching the levels observed in ChAdOx1 nCoV-19 induced VITT patients. Moreover,
H3Cit, a marker of NET generations, the HPV vaccinated had lower or similar levels than
the VITT patients and controls, respectively, suggesting lack of NET formation in the HPV
vaccinated patient.

Whereas the levels of all the tested markers were markedly increased during the acute

36
complication in the HPV vaccinated patients as compared with levels four months prior to
vaccination, there was a full normalization of all levels at 12 months follow-up.

The first reports of VITT patients raised the awareness of the conditions catalyzed by
vaccine-induced immune response. Thus, a few months after the reports of the VITT
patients, a hospitalized young woman developed thrombocytopenia, thrombosis and
platelet-activating antibodies to PF4 – complex in Norway after receiving the Gardasil
vaccine [1]. Although perhaps not surprisingly, this data show a moderate and transient
increase in a wide spectrum of inflammatory related markers following HPV vaccination,
that to the best of our knowledge, has not previously been reported. These responses
include markers of vascular inflammation, macrophage activation and some degree of
neutrophil activation, and notably, sCD163 level as a marker of M2 macrophage activation
was even higher than in the VITT patients. Unlike ChAdOx1 nCoV-19, Gardasil is not an
adenovirus vector-based vaccine, but a virus capsid preparation. Caution is clearly needed
when interpreting data from only one patient, it may, however, seem likely that the
immune reaction upon vaccination rather than the vehicle or antigen itself may be causal.
These data should be taken into consideration when next generation vaccines are designed.
The present findings should further strengthen the clinical awareness of vaccine-induced
complications which may include immune activation and inflammation along with
thrombus formation as a potential common feature.
3.Thrombocytosis has been frequently found in association with cancer.
Levin and Conley found that 0.6% of 14,000 adult inpatients at The Johns Hopkins
Hospital (Baltimore, MD) had thrombocytosis (platelet count > 400,000/~1) identified on
blood smear and verified by platelet count. Thirty-one (38%) of the 82 patients with
thrombocytosis in their study had cancer. They also studied 268 consecutive patients
referred for chemotherapeutic treatment of inoperable malignancies.

Forty percent of these patients (excluding those with carcinoma of the lung) had
thrombocytosis. Among patients with carcinoma of the lung, thrombocytosis was identified
in 38% of the cases. A study of 190 patients with lung cancer by Silvis et al. found that 60%
of their patients had thrombocytosis. Of the113 patients treated with radiation therapy for
cervical cancer, 20 (1 7.7%) had thrombocytosis. Even though thrombocytosis may be
found in association with a number of disease processes, in this series the patients with
thrombocytosis did not differ in the number or types of chronic illnesses when compared
with those with normal platelet counts. Thrombocytosis may occur from an infectious
etiology, but we found no difference in the leukocyte count between patients with
thrombocytosis and those with normal platelet counts.

The specific mechanisms by which thrombocytosis is produced in cancer patients are

unknown; however, there have been several hypotheses proposed on this subject. The
elevated platelet count does not seem to be due to an increased life span, rather there is an
increased production of thrombocytes by the megakaryocytes.' There is evidence that the
tumor cells secrete stimulating factors which will eventually lead to thrombocytosis.
Thrombocytosis is seen in patients with severe iron deficiency anemia." Even though the

37
patientsthe patients with thrombocytosis had a lower hemoglobin than patients with normal
platelet counts, all but four had hemoglobin levels of 10 mg/dl or greater. The remaining
four had hemoglobin levels between 8 and 9 mg/dl.

Thrombocytosis may worsen the prognosis for patients with cancer due to the interactions
between the platelets and the cancer cells. An increase in platelets may promote cancer cell
attachment, which is the first step in metastasis. Thrombospondin, a platelet-secreted
protein, has been shown to potentiate tumor cell metastasis in an animal model." It is
proposed that thrombospondin promotes the adhesion of tumor cells to the endothelium.
The effect of thrombospondin on metastasis is dependent on the presence of platelets and a
normal clotting system. '' In the clinicsthe clinics, high circulating levels of thrombospondin
were found among patients with metastatic disease."

There is also evidence that the platelets protect the tumor cells by shielding them from the
humoral and cellular defense mechanisms present in the host. The tumor cells, with
platelets attached, may lodge more readily in a microvascular bed which contains the
platelet-specific binding sites. Only 0.05% or less of the tumor cells in the bloodstream are
able to escape host's defense mechanisms, implant on vascular endothelium, and
invade.Anyinvade. Any aid from the platelets in this matter will surely improve the odds for
metastasis to become established, thereby resulting in a poorer prognosis for patients with
both thrombocytosis and cancer.

Studies in animal models have shown that platelet reduction prevents metatasis. In the
analysis foundanalysis found thrombocytosis to be a prognosticator of poorer survival
independent of patient's age, disease stage, or histologic type. This supports the theory that
thrombocytosis aids in the establishment of metastasis. Conversely, thrombocytosis may be
a marker for occult advanced disease with the thrombocytosis being the result of
substances produced by the larger tumor burden. Regardless of which of these two
hypotheses is correct, thrombocytosis is a poor prognostic indicator in cervical cancer.

C. A Case Study Result Indicating Leukocyte Changes in Cancer Patients.

Leukocytosis
Pretreatment leukocytosis according to the established criteria was observed in 35 (11.9%)
patients before the initiation of the chemoradiation treatment. The median white blood cell
count (WBC) in patients with leukocytes at their first visit was 13300/µL (11100-28800).
According to the differential leukocyte counts, in most cases there was a clear
predominance of granulocytes with percentage ranging from 65.3% to 91.1% (median
77.4%). The median absolute granulocyte count was 10300/µL with range from 7300 and
25630. Before initiating chemoradiation there were not major changes in leukocyte
numbers between at least three separate counts taken before commencing chemoradiation
(means: 14458, 15879, 15297; medians: 13300, 14650, 13300/µL).

Patient characteristics

38
 The mean age of the group with leukocytosis was 47.8 years (range, 29-78 years).
Histologically, there were 32 squamous cell carcinomas (91%), one adenosquamous
carcinoma (3%) and two with other histologies (6%). The distribution of the clinical stages
according to the FIGO classification of these patients was: four patients had stage IB2
(11%), four IIA (11%), 12, IIB (35%), 13, IIIB (37%) and two (6%) had IVA stage. The
mean hemoglobin level at diagnosis was 11.8 g/dL with ranges between 6.4 and 15.7 g/dL.

Correlation between leukocytosis and clinicopathological factors

Associations between the presence or absence of leukocytosis and patient
clinicopathological showed that there was only statistically significant association between
leukocytosis and the stage of the disease. Patients with leukocytosis presented a more
advanced stage of disease (III and IVA) than the patients who did not have leukocytosis.
Leukocytosis was unrelated to age, hemoglobin level and tumor histology.

Correlation between leukocytosis and response to chemoradiation

There was a strong statistically significant association between the presence or absence of
leukocytosis and the clinical response. Eighty-six percent (223/259) of the patients with no
leukocytosis achieved complete response as comparison to only 57% (20/35) for those
having leukocytosis. Accordingly, 31.5% and 11.5% of leukocytosis patients presented
persistent and progressive disease respectively versus 8% and 6% for those with no
leukocytosis (p<0.001).

Leukocytosis and survival

At a median follow-up 28 (2-68) months, overall survival for the whole group is 76.5%. The
analysis of survival according to the presence of leukocytosis shows that the median
survival time was 22.2 months (5-58) and 25.1 (2-68) for those with and without
leukocytosis respectively, p=0.0036. The Cox proportional hazards model was used to
evaluate the effect of leukocytosis on patient survival while adjusting for clinical and
pathologic prognostic factors. These included the presence or absence of leukocytosis, age,
stage of disease, histology and hemoglobin levels. Age was grouped into two categories,
younger and older than 35 years; disease stages into IB2-IIB, versus III-IVA; histology was
grouped into squamous versus adenocarcinoma, adenosquamous and others, whereas
hemoglobin between >10 gr/dL or <10 gr/dL. In the univariate analysis leukocytosis, the
stage of the disease and the hemoglobin levels were significant predictors of survival
however, only leukocytosis and the hemoglobin level remained significant predictors of
survival in the multivariate analysis .
Leukocytosis and Prognosis in Cervical Cancer
Several authors have indicated a correlation between leukocytosis and poor prognosis in
cervical cancer. Okazawa-Sakai et al. , in a population of 219 patients with FIGO stage
IIB–IVA cervical cancer, demonstrated that pretreatment leukocytosis and pretreatment
neutrophilia were significant independent predictors of distant relapse. Maulard et
al. ,inal., in 238 patients with locally advanced cervical cancer, demonstrated that
leukocytosis, together with anemia and thrombocytosis, was correlated with reduced
disease-free survival and overall survival. Koulis et al. have reported that leukocytosis and
increased NLR,together with anemia, were associated with worsened PFS and OS in 257

39
patients with cervical cancer treated with radical chemoradiotherapy . Similarly, Garcia-
Arias et al.,in a retrospective analysis of 294 consecutive patients with new diagnosis of
untreated locally advanced cervical cancer, demonstrated that leukocytosis and
hemoglobin level were predictive of poor outcome . Mabuchi et al. revealed in a
retrospective analysis of 536 patients with cervical cancer that leukocytosis at the time of
recurrence was an independent prognostic factor and correlated with short survival after
recurrence (median:9 months).

Others have reported on more general hematologic markers related to inflammation such
as neutrophilia and elevated neutrophil-to-lymphocyte ratio (NLR) . As the inflammation
process is linked with cancer growth, different circulating inflammatory markers have
been evaluated for their potential role as prognostic and predictive factors of clinical
outcomes in different cancer types. The NLR is calculated from the ratio between the
neutrophil and lymphocyte counts obtained from a full blood count. It has been suggested
as one of the most helpful prognostic indices in cancer . In patients with cervical cancer, the
NLR has been reported as a poor prognostic factor in addition to tumor stage.

Some clinical studies have reported the correlation between pre-treatment NLR and the
prognosis of cervical cancer . Meanwhile, more recently, have demonstrated in a
population of patients with ovarian cancer that the pretreatment NLR value does not
constitute a prognostic and predictive factor, but only the evidence of the extent of the
systemic inflammation in progress; conversely, the increase in the number of lymphocytes
during chemotherapy treatment, and therefore the reduction of NLR, constitutes one of the
most valid predictors of response to treatment .

3.5 PROTECTION AGAINST CANCER

AsCANCER As a result of its unique mechanisms of infection, contact with high-risk HPV
types 16 and 18 most often lead to cancer development. These papillomaviruses are the
only known viral infections that self-initiate, interacting with the cell surface when it
attacks. After that, it takes a mere 24 hours for HPV to fully assimilate the basal cell’s
nuclear DNA with its own. As a result, the transcription and translation of the infectious
genetic material leads to the production of viral proteins that process the disease. Types 16
and 18 express proteins (L1 and L2) not found in low-risk HPV types that are diametrically
linked to oncogenesis (cancer cell production) and the promotions of invaded skin cell
growth. L1 and L2 contribute to HPV’s “outer cap layer” or “capsid layer” binding to the
basal membrane, the basal cells of future hosts, and the base of the tongue and oral
pharynx as with cervical cells.

Cervarix and Gardasil vaccines were used to prompt the recognition of and the antibody
protection of a person’s natural immunity toward the L1 capsid protein. However, in
accordance with the varied expressions of L1, defense is limited by the diminishing amount
of cross-type protection from these kinds of vaccines. Cervarix and Gardasil only induce
immunity against types 16 and 18 and types 6, 11, 16, and 18 respectively. Their antibody

40
responses plateau 12–18 days after injection and decline until the subsequent injection in
the series.

With the help of statistical models, it is predicted that the immune response to L1
antibodies have sufficient memory to recognize and therefore respond to future HPV
challenges for 32 years, saving boys and men from the ravages of cancer long after the
vaccination series is completed.Currentcompleted. Current evidence even suggests that the
immune memory of L1 induced by the vaccine remains strong enough up to 7 years
afterward that it can reliably respond to new forms of HPV.

While 16 and 18 may be the most commonly occurring HPV genotypes, international
distribution varies greatly, accounting for 79% of carcinomas in North America and 68%
of carcinomas in Africa. As such, a second generation of HPV vaccines, including Gardasil
9 are currently being developed with the intent of providing broader crosstype immunity,
targeting L2 as it is more highly conserved (providing coverage) across HPV types, and
most of all to cover more than just HPV types 6, 11, 16, and 18.

Research experts, including those from the Centers for Disease Control (CDC), have
reported that HPV infections are increasing across cancer sites but remain hopeful in light
of the supported increase for vaccination efforts.With HPV-related cancer on the rise, it
becomes more and more apparent that preventing these cancers through the completion of
the prophylactic HPV vaccination series is vital, especially for adolescent boys and girls.

Description of the Vaccine and Current Utilization

Because it is such an effective method of prevention, the HPV vaccine will always be a
crucial health promotion element for both boys and girls alike.24 A simple series of 3
injections over a period of 6 months is an essential step towards reducing HPV-related
cancer.Unfortunately, rates of vaccination completion continue to be low, 40% nationwide
for girls ages 13 to 17 with Florida having the lowest rate completion rate for both boys
(17%) and girls (28%) in the nation.With those kinds of figures, a simple series of 3
injections over a period of 6 months is an essential step towards reducing HPV-related
cancer.

Vaccination has been approved by the Advisory Council on Immunization Practices

(ACIP), and the CDC to prevent HPV-related cancers including head, neck, throat,
cervical, and other cancers caused by HPV types 6, 11, 16, and 18 (subtypes responsible for
approximately 99% of cervical cancers and 90% of genital warts) is recommended for girls
and boys ages 9 to 17. In 2009, the HPV vaccine was approved by the Food and Drug
Administration (FDA) for use in males ages 9 to 26.Two years later, ACIP and the CDC
recommended using quadrivalent vaccines in boys aged 11–12, catch-up vaccines in boys
ages 13–21, and males ages 22–26 (recommendation for routine use not given).

In February 2015, a 9-valent HPV vaccine called Gardasil 9 was approved for use in both
males and females by the FDA.20,28 Licensed for use in males ages 9–15 and females 9–26,

41
this new vaccine was designed to guard against types 6, 11, 16, and 18 with additional
protections against types 31, 33, 45, 52, and 58. Gardasil 9 not only received full committee
approval from ACIP but it was agreed its 97% efficacy rate and ability to protect against
HPV-related cancers was superior to Gardasil for both males and females. And yet, with no
cited preferences for one vaccine or the other it is certain that HPV vaccinations will be a
conversation between healthcare providers and their patients.
.
Even with all of its recommendations and the demonstrated safety and efficacy, HPV
uptake remains low. HPV immunization completion rates stay well below projected figures
in males (less than 21%), females (less than 60%).These low vaccine rates translate to 79
million people currently infected and another 14 million more people newly infected every
year in the U.S. While such alarming rates of cancer continue to rise, completing the
prophylactic HPV vaccine series is direly important, especially concerning adolescents in
areas of poverty, low literacy, and limited access to primary care and prevention services.

Luckily, the concern over mounting HPV-related cancer rates is becoming the subject of
more and more studies and even the President’s Cancer Panel (PCP) in 2014 made the
issue a priority of focus and concern for 2015.

CHAPTER FOUR

4.1. CONCLUSION
This study comprehensively investigated the dynamic alterations in leukocyte and
thrombocyte counts throughout the course of cervical cancer treatment. The findings
highlight the crucial role of hematological parameters in assessing disease progression,
predicting treatment response, and ultimately improving patient outcomes. The intricate
interplay between high-risk HPV infections, the oncogenic mechanisms of E6 and E7

42
proteins, and the resulting immune dysregulation and hematological disorders has been
elucidated.
Advanced diagnostic techniques, particularly PCR and RNA-based assays, are
instrumental in early detection and prediction of disease progression, underscoring the
need for integrating these methods into routine clinical practice. Furthermore, the
research emphasizes the importance of routine hematological monitoring during and after
treatment to guide therapeutic strategies, enhance treatment efficacy, and ultimately
improve patient quality of life.
The integration of comprehensive hematological assessments into cervical cancer
management is crucial for reducing the global burden of this significant public health
challenge.
Further research should focus on the identification of novel biomarkers and the
development of targeted therapies to improve patient outcomes and reduce morbidity and
mortality associated with cervical cancer.

4.2 RECOMMENDATIONS
Cervical cancer treatment has significant effects on thrombocytes and leukocytes, leading
to potential hematological complications. To mitigate these effects and improve patient
outcomes, the following recommendations are proposed:
1. Regular Hematological Monitoring: Patients undergoing cervical cancer treatment
should receive regular hematological monitoring, including complete blood counts (CBC)
and platelet counts, to detect early changes in thrombocytes and leukocytes.
2. Personalized Treatment Planning: Treatment plans should be tailored to individual
patients' hematological profiles, taking into account potential risks and complications
associated with thrombocytopenia and leukopenia.
3. Platelet and Leukocyte Supportive Care: Patients experiencing thrombocytopenia or
leukopenia during treatment should receive supportive care, including:
- Platelet transfusions as needed
- Granulocyte-colony stimulating factor (G-CSF) administration to stimulate leukocyte
production
- Antibiotic prophylaxis to prevent infections
4. Patient Education and Awareness: Patients should be educated on the potential
hematological effects of cervical cancer treatment and encouraged to report any symptoms
or concerns to their healthcare providers, including:
- Bleeding or bruising
- Fatigue or weakness
- Fever or infection
- Shortness of breath or chest pain

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5. Multidisciplinary Care: A multidisciplinary approach to care, involving hematologists,
oncologists, gynecologists, and other healthcare professionals, is essential for optimizing
patient outcomes and managing hematological complications.
6. Dietary and Lifestyle Modifications: Patients should be advised on dietary and lifestyle
modifications to support hematological health, including:
- A balanced diet rich in iron, folate, and vitamins
- Adequate hydration and rest
- Avoidance of smoking and excessive alcohol consumption
7. Psychological Support: Patients should have access to psychological support and
counseling to manage the emotional and psychological impacts of cervical cancer
treatment.
8. Future Research Directions: Further research is needed to investigate the hematological
effects of cervical cancer treatment and to develop more effective strategies for mitigating
these effects.
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