Nuclear Medicine Resources Manual
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CHAPTER 5. GUIDELINES FOR GENERAL IMAGING
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Day 1:
—Dispense 0.5 g of magnetic cellulose into a polystyrene tube.
—Add 0.5 mL of 10 mg/mL IgG solution in a barbitone buffer of pH8 and
adjust the volume to 10 mL with a bicarbonate buffer of pH8.
—Leave the tube rolling overnight at room temperature.
Day 2:
—Sediment the particles and wash twice with 20 mL bicarbonate buffer.
—Sediment the particles and wash with 20 mL bicarbonate buffer
containing 3
mL/L ethanolamine.
—Sediment the particles and resuspend in 20 mL of bicarbonate buffer
containing 3 mL/L ethanolamine and roll for 30 min at room temperature.
—Sediment the particles and resuspend in 20 mL acetate buffer of pH4 and
roll for 30 min at room temperature.
—Sediment the particles and wash twice with 20 mL assay buffer.
Day 3:
—Pipette 5, 10, 20, 50, 100 and 200 mL magnetic cellulose suspensions in
duplicate into assay tubes (adding assay buffer for constant volume).
—Add 100 mL labelled rabbit IgG solution and leave at room temperature
for two hours, with occasional shaking to keep the particles in suspension.
—Add 1 mL wash solution; sediment the particles and aspirate the super-
natant. Wash the particles with a further 1 mL wash solution and count
the magnetic pellets.
—Plot a dilution curve for the solid phase antibodies.
5.13. MOLECULAR METHODS — USE OF RADIONUCLIDES IN
MOLECULAR BIOLOGY
5.13.1. Introduction
Cell anatomy can be studied by several distinct methods using
microscopy but, although revealing cellular architecture, very little information
is provided about cell physiology. The biochemistry and molecular biology
perspective is to identify molecules involved in a specific pathway and to
determine where and why the pathway occurs. To address these points, it is
5.13. MOLECULAR METHODS
411
necessary to deal with a spectrum of different molecules, ranging from
inorganic ions to large macromolecules such as nucleic acids and proteins.
In the 1970s, several technical approaches were described bringing
sensitive methodological advantages that drastically altered the amount of data
generated in this field of research. To monitor the molecular aspects of cell
physiology, radioisotopes are routinely used, for example, in tracing chemical
pathways, evaluating the dynamic behaviour of compounds in cytosol and
nucleus, and identifying and typing molecules. Radioisotopic methods are
specific and sensitive, capable of detecting a particular molecule sample
present in small amounts in complex mixtures.
In human health, molecular biology has several applications. The most
developed are in the molecular diagnosis of infections, genetic diseases and
cancer. Molecular techniques can be applied to the diagnosis of diseases such as
human papilloma virus infection and Chagas’ disease (infective diseases),
Fragile X syndrome (a genetic disease) and the mutated p53 gene (cancer).
Besides improving diagnosis, molecular methods can also be used to
address the control of disease through:
—Identification of common transmission sources;
—Assessment of drug resistance;
—Follow-up of treatment efficacy;
—Strain typing to distinguish more pathogenic organisms.
5.13.1.1. Molecular diagnosis
The polymerase chain reaction (PCR) is an in vitro method of nucleic
acid amplification by which a particular segment of DNA can be specifically
replicated. It involves two oligonucleotide primers that flank the DNA
fragment to be amplified, repeated cycles of heat denaturation of the DNA,
annealing of the primers to their complementary sequences and extension of
the annealed primers with DNA polymerase. The combination of the four
bases that constitute the DNA (adenine, thymine, cytosine and guanine) in an
approximately 20 base segment, which corresponds to the oligonucleotide
primers, is unique enough to be specific to a single microorganism or a
mutation. This specificity is combined with the sensitivity of the PCR due to the
exponential amplification of the target. Specificity is further enhanced by
molecular hybridization using probes, making these approaches ideal tools for
diagnostic purposes.
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5.13.2. Molecular diagnosis of infective diseases using molecular techniques
(PCR coupled with hybridization with radioactive probes)
Molecular approaches allow ready detection (in one or two days) of
single pathogenic organisms, an accomplishment provided before by in vitro
culturing (requiring weeks) of such pathogens. Furthermore, they allow
detection of pathogens, such as the human papilloma virus, that are refractory
to in vitro propagation. Antibodies used in a direct search for a given pathogen
typically recognize antigens found in multiple copies on the microorganism and
thus circumvent the need to replicate the agent. Unfortunately, the cross-
reactivity of these antibodies with host antigens and other pathogens has
compromised the convenient and broad use of these diagnostic reagents for
some pathogens. In addition, some viruses establish latent infections in which
active viral replication is substantially attenuated, thereby preventing detection
by antigen based methods.
Molecular diagnostic methods can provide non-invasive alternatives. For
example, in visceral Leishmaniasis, the parasitological diagnosis can be
performed using peripheral blood instead of bone marrow or spleen aspirates.
Similarly, in chlamydia infections, urine can be used instead of urethral
scrapings.
In Chagas’ disease, parasitological diagnosis can be performed by PCR
and molecular hybridization using blood. This approach is in contrast to the
traditional xenodiagnosis method, where 40 insects starving for 45 days are
applied to the patient’s arms. In cutaneous Leishmaniasis, the invasiveness is
reduced from regular biopsies to needle aspirates.
Examples of pathogens that can be detected using these approaches are
listed below. Pathogens with an asterisk represent those for which molecular
based methods are the gold standard:
Adenovirus Legionella pneumophila
Bartonella henselae and Bartonella quintana * Leishmania sp.
Borrelia burgdorferi Microsporidia
* Chlamydia pneumoniae Mycobacterium avium
* Chlamydia trachomatis Mycobacterium bovis
Cytomegalovirus Mycobacterium leprae
Epstein–Barr virus Mycobacterium tuberculosis
Helicobacter pylori Mycobacterium ulcerans
Hepatitis B virus Mycoplasma pneumoniae
* Hepatitis C virus * Neisseria gonorrhoeae
Hepatitis G virus Onchocerca volvulus
Herpes viruses 6 and 8 Parvovirus B19
5.13. MOLECULAR METHODS
413
Herpes simplex types 1 and 2 Plasmodium sp.
Histoplasma capsulatum Pneumocystis carinii
* HIV Toxoplasma gondii
HTLV I/II Trypanosoma cruzi
* human papilloma virus Varicella–Zoster virus.
In this section, two distinct models are chosen for more detailed consider-
ation: human papilloma virus (HPV) infection and Chagas’ disease.
5.13.2.1. Human papilloma virus
HPV is the most common genital virus, with transmission mediated by
direct contact. More than 70 genotypes based on DNA sequence have been
described and clustered into subtypes. The virus site of latency is the
epithelium, where most of the clinical presentations are encountered. The
distinct virus genetic groups present different cellular tropisms and therefore
present distinct clinical features (skin warts, benign head and neck tumours,
genital warts and cervical carcinoma). For example, HPV 1 produces plantar
warts, HPV 2 and 4 cause common warts on the hands, HPV 6 and 11 are
involved with genital warts and HPV 16 and 18 are the causative agent of
genital warts and cervical carcinoma.
The possibility of detecting HPV in a clinical sample depends on the
sensitivity of the assay. No culturing system is available. Histopathological or
cytological analyses are indirect methods detecting morphological alterations
produced by HPV. The presence of ‘HPV changes’ or koilocytes is neither
sensitive nor specific to the presence of the virus and cannot differentiate
between high and low risk subtypes. Identifying the distinct HPV subtypes is
possible due to genetic differences that are present in the different viruses.
Molecular methods have been developed in order to detect and type the virus
in clinical specimens. This is achieved by molecular hybridization with radio
-
labelled probes in the Southern Blot procedures.
Ninety-five per cent of viruses that belong in the low risk group are typed
as HPV 6, 11, 42, 43 and 44 and cause anogenital warts. The high risk group is
associated with the development of cervical intraepithelial neoplasia (CIN).
Ninety-nine per cent of the viruses of this group can be typed using probes to
HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. HPV DNA is detected in
95–100% of cervical tumour samples and is considered as the major risk factor
for cervical cancer. Fifteen–28% of HPV positive women will develop
neoplasia within 2 years in contrast to 1–3% of HPV negative women.
Therefore, the relative risk of developing CIN is 11 times greater in HPV
positive women.
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HPV interaction with host tumour suppressor proteins, such as p53, is the
oncogenic basis for the development of cancer. Polymorphism in the host p53
gene has prognostic significance, defining groups that are more susceptible to
cancer development and therefore requiring closer follow-up, leading to early
detection and treatment of cervical cancer.
Considering of the high prevalence of cervical cancer and its association
with HPV, a molecular approach that detects and types the virus, determines
the viral load and analyses the oncoprotein involvement is helpful in
identifying the risk group of women who need close follow-up. These molecular
methods are complementary to conventional investigations such as cervical
smears and histopathological analyses (see the chronology of investigation in
Fig. 5.4).
5.13.2.2. Trypanosoma cruzi and Chagas’ disease
Chagas’ disease is caused by the protozoan T. cruzi, which is mainly
transmitted by an insect vector, the triatomine bug. The infective stages of the
parasite are present in insect faeces and can penetrate through the epithelium
into host cells. During the acute phase, which can last up to 60 days, a large
number of circulating parasites are observed in the bloodstream. Patients
may develop different clinical presentations, varying from oligosymptomatic
to the development of myocarditis and meningoencephalitis. Following the
acute phase, a chronic stage develops in which the level of circulating
parasites is far below the threshold for microscopic detection. Patients are
symptomless for several years with the only marker for the T. cruzi infection
being the high titre of anti-T. cruzi antibodies. Approximately 10–20% of
infected individuals will develop a symptomatic chronic disease hallmarked
by cardiovascular–gastrointestinal involvement.
In addition to transmission through the insect vector, T. cruzi may also be
transmitted congenitally or by blood transfusion from asymptomatic carriers.
The infected newborn may show a clinical picture characterized by prematurity,
hepatomegaly, splenomegaly, jaundice, anaemia and alterations in the CNS,
resulting in a high mortality. Chagas’ disease due to blood transfusion is a
problem in Latin America since in some endemic areas a significant percentage
of potential blood donors (2–63%) may be infected. In the last few years,
reactivation of Chagas’ disease has been reported, mainly associated with
immunosuppression in hosts with HIV infection and individuals undergoing
organ transplantation.
Etiological therapy for Chagas’ disease is indicated in either acute or
recent chronic infections of less than ten years that seem to respond to
treatment. In practice, it is recommended that all children with positive
5.13. MOLECULAR METHODS
415
BOTH
Repeat PAP and HPV at 6 months
HPV POSITIVE OR HIGH GRADE
ALTERATIONS
PAP SMEAR MINOR ALTERATIONS = REPEAT
TEST AT 6 MONTHS AND SAMPLE FOR HPV
HPV NEGATIVE + LOW GRADE
ALTERATIONS
Colposcopy
Re
p
eat PA
P
BOTH
Follow with PAP smear at
2
y
ear intervals
Lesion
No lesion -
DNA carrier
Treat
Follow with PAP smear at 6
month intervals for 4 years
Either or
both
POSITIVE
Both
NEGATIVE
FIG. 5.4. Flow chart showing the chronology of an investigation for HPV.
CHAPTER 5. GUIDELINES FOR GENERAL IMAGING
416
serological reactions be submitted for specific therapy. Furthermore, patients
with the chronic indeterminate form of the disease, the slight cardiac–digestive
form, should also be treated.
The diagnosis of Chagas’ disease can be made by different means
depending on the phase of the disease. In the initial phase of T. cruzi infection,
large numbers of parasites circulate in the blood, and diagnosis is primarily by
direct microscopy. By contrast, in the chronic phase, circulating levels of
parasites are low and therefore diagnosis depends on detection of the host
serological response or on in vitro amplification of the parasites, such as
xenodiagnosis or haemoculture. In the former, 40 uninfected triatomine bugs
are allowed to feed on the patient and a month later the intestinal contents of
the insects are examined for the presence of T. cruzi. However, both parasito
-
logical methods lack sensitivity, and positive findings are achieved in less than
50% of seropositive chronically ill patients. In addition, these methods may
select parasite subpopulations, distorting the typing of the involved parasite
and epidemiological data.
Although conventional serological assays can offer fast and fairly reliable
diagnosis, they lack specificity, giving rise to false positive results that need
confirmation by a parasitological test. Moreover, in congenital infection,
serology is precluded by the circulation of maternal IgG antibodies during the
first six months of life. The early diagnosis of congenital transmission is
essential because treatment is more efficient when given closer to the time of
delivery. Thus, a highly sensitive parasitological assay is needed for the
diagnosis of an infected newborn of a Chagasic mother or for monitoring the
presence of the parasites in the chronic phase of the disease.
Oligonucleotides derived from the conserved region of mitochondrial
DNA have been used in PCR based assays to detect this parasite in human
blood samples. The amplified products are detected by gel electrophoresis and
hybridized with a radiolabelled molecular probe. The PCR assay can attain a
better sensitivity and specificity than combined serology and clinical diagnosis.
Furthermore, PCR positivity is much better than any other direct parasito
-
logical method, such as xenodiagnosis and haemoculture. Details of the
appropriate use of the molecular diagnosis of Chagas’ disease are given in
Fig.
5.5.
5.13.3. Molecular diagnosis of genetic diseases using radioactive labelling
It is known that several human diseases are caused by defective genes, but
until very recently very few had been identified. The identification of such
genes for a number of important diseases, such as cystic fibrosis, Huntington’s
disease, Fragile X syndrome and haematological disorders, has led to the
5.13. MOLECULAR METHODS
417
+
CHAGAS’ DISEASE
+
CHAGAS’ DISEASE
Presence of the parasite
PCR
Negative
Wet smear
Stained smear
Strout method
(direct search for the parasite)
Interpretation of specific
Serology:
situations is required
ACUTE
No Chagas’ disease
Both negative
Absence:
No Chagas’ disease
+
CHAGAS’ DISEASE:
Presence of the parasite
PCR
Presence
Clinical signs
and symptoms/
Epidemiological evidence
Doubtful result
- +
Confirmatory parasitological
test
+ +
CHAGAS’ DISEASE
Serology
performed by at least two distinct
methods following WHO
recommendations
CHRONIC
CLINICAL SIGNS OR SYMPTOMS AND/OR
EPIDEMIOLOGICAL EVIDENCE OF CHAGAS’ DISEASE
FIG. 5.5. Flow chart showing chronology of investigation for molecular diagnosis of Chagas’ disease.
CHAPTER 5. GUIDELINES FOR GENERAL IMAGING
418
application of molecular methods to the diagnosis of such diseases. Hundreds
of mutations can cause the same genetic disease. For example in b-thalassaemia
approximately 200 mutations are implicated. However, for any particular
ethnic group, about ten mutations will cover 90% of the genotype spectrum,
simplifying screening strategies in genetic programmes. PCR technology has
further simplified prenatal diagnosis and carrier testing.
Molecular genetics laboratories now offer tests for a large number of
diseases, such as:
a- and b-Thalassaemia Huntington’s disease
Alport’s syndrome Myotonic dystrophy
Alzheimer’s disease Niemann–Pick’s disease
Androgen’s insensitivity Ornithine transcarbamylase deficiency
Angelman’s syndrome Prader–Willi’s disease
Antitrypsin deficiency Retinitis pigmentosa
Charcot–Marie–Tooth’s disease Sickle cell anaemia
Choroideraemia Spinal muscular atrophy
Cystic fibrosis Tay–Sach’s disease
Fragile X syndrome Von Hippel–Lindau’s disease
Haemophilia A Von Willebrand’s disease
Hereditary neuropathies X-linked muscular dystrophy.
In this section, a model of a genetic disease, Fragile X syndrome, in which
the molecular diagnosis has had an impact in prevention, is given in more
detail.
5.13.3.1. Fragile X syndrome
Fragile X syndrome (also termed FRAXA) is the most common form of
inherited mental retardation in humans, and is associated with a wide range of
behavioural and physical features, affecting both males and females, with a
prevalence of 1 in 4000 males and 1 in 6000 females. The cloning of the FMR1
(Fragile X mental retardation 1) gene and the identification of the mutational
mechanism underlying the Fragile X syndrome, a large expansion of a
trinucleotide repeat (CGG)
n
in the first exon of the FMR1 gene, has allowed
the development of accurate molecular diagnostic tests, which have replaced
cytogenetic marker analysis (of a fragile site at Xq 27.3) as the procedure of
choice and dramatically improved diagnosis of the disease.
The standard diagnostic procedure used is to measure the CGG repeat
amplification in patients. More than 99% of Fragile X syndrome cases
identified have resulted from CGG expansions, with only a few cases derived
5.13. MOLECULAR METHODS
419
from FMR1 point mutation or deletions. Since the great majority of Fragile X
patients share a mutation at precisely the same site in the gene, as opposed to a
range of mutations scattered along the length of a gene, the genetic diagnosis
(or exclusion) of Fragile X syndrome is remarkably reliable.
Southern Blot analysis is the procedure of choice for medical diagnosis of
Fragile X syndrome. The use of this methodology is essential to characterize
the Fragile X mutation in males and females, to distinguish premutation from
full mutation and to detect methylation.
The probes frequently used to identify CGG amplification are StB12.3,
Pfxa3 and Ox1.9. For Southern Blot analysis, different restriction enzymes may
be used, such as PstI and EcoRI. A double digest, including a methylation
sensitive restriction enzyme (EagI, SacII and BssHII), enables detection of the
methylation of the CGG repeats and associated CpG island. The double digest
may also be used in distinguishing between an unmethylated large premutation
and a small methylated full mutation. The EcoRI/EagI protocol using the
Stb12.3 probe is a standard diagnostic procedure. The digestion of genomic
DNA with EcoRI (insensitive) and EagI (sensitive), after hybridization with
the StB12.3 probe radiolabelled with [a-P 32]dNTP, generates a 2.8 kb EcoRI–
EagI fragment from a normal unmethylated allele (representing an inactive X
chromosome). Southern Blot methods allow the molecular classification of
alleles by estimation of the approximate size of the Fragile X expansion.
Premutation alleles are detected as 5.3–5.7 kb EcoRI–EagI fragments, whereas
full mutation alleles, which are consistently methylated and thus not cleaved by
EagI, are detected as EcoRI fragments larger than 5.8 kb in size. The ability to
discriminate between a premutation and a full mutation in the FMR1 gene for
the use of this methodology provides a much improved prediction of the risk of
mental retardation.
Because of the prevalence, underdiagnosis and high risk of recurrence of
Fragile X syndrome, direct DNA analysis of the FMR1 gene is extremely
important in providing correct diagnosis of the disease and may be used in the
differential diagnosis of individuals with mental retardation of unknown
aetiology and in the genetic counselling of families with Fragile X syndrome.
5.13.4. Molecular diagnosis of cancer
Cancer is due to genetic alterations that affect cell growth and differenti-
ation. The molecular approach to cancer aims at three distinct goals:
(1) Identification of mutations that predispose an individual to cancer
development (BRCA1 in breast cancer);
(2) Detection of minimal residual disease (following leukaemia treatment);