Complete Lecture Notes Clinical Laboratory Sciences Cls PDF

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A booklet of all CLS revision and lecture notes....

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CLS1: The Study of Disease List the disciplines of clinical laboratory studies: Pathology - the study of patterns, causes, mechanisms and effects of illness (disease). Histopathology - the study of the microscopic anatomical changes in diseased tissue. Haematology – the analysis of blood (+ other fluids) Clinical chemistry - the science involving chemical analysis of body tissues to diagnose disease Immunology - studies the body's immune system Microbiology - the isolation and characterisation of infective agents (micro-organisms) Genetics - genetic causes of disease Define the terms aetiology, pathogenesis, natural history, sequelae and prognosis: 1. AETIOLOGY – the root cause of a disease - Surgical sieve (VITAMIN D – vascular, infection, trauma, autoimmune, metabolic, inflammation, neoplasia, degeneration) 2. PATHOGENESIS – the development of a disease or morbid condition. Very important in choosing, or developing, an appropriate therapy. 3. NATURAL HISTORY – the course of a disease process. Affected by being untreated or modified by therapy. 4. SEQUELAE – the clinical and pathological consequences of a disease process i.e. the knock on effect 5. PROGNOSIS - a prediction of the probable course and outcome of a disease. (i.e. either survival, disease-free survival or disability) Outline the main causes of disease and describe relevant examples of each: Congenital disease – may be developmental, genetic, sporadic Genetic disease – onset at any age by a variety of genetic mechanisms Metabolic disease – disorder that involves an alteration in the normal metabolism, can be genetic or acquired Inflammatory disease – usually secondary to tissue damage (itis conditions) Neoplastic disease – tumours, malignant carcinomas, benign tumours Environmental disease – physical, chemical or radiation e.g. burns

Immune disease – autoimmune or immune deficiency Infective disease – could be a virus, bacteria, fungi, protozoa or parasites Vascular disease - disorders that involve the arteries, veins, and lymphatics. e.g. atheroma Degenerative disease – wear and tear of organs and joints with age Iatrogenic disease - Induced in a patient by a physician's activity, manner, or therapy. e.g. drug side effects. (20% made worse by doctor)

Give a definition of the term disease: Disease: “An impairment of health or a condition of abnormal functioning” Normal: the normal range for whatever is set to cover 95% of all values from the general population. The normal range may differ depending on the patient's age, size, sex, or ethnic background. Outline the autopsy procedure and its purpose: What? – an examination of a cadaver to determine or confirm the cause of death. (postmortem) Why? - under law (coroner) or under the human tissue act (having given consent). 30% of all death certificates have incorrect cause of death. How? – carry out both external and internal examination, remove and dissect organs.

CLS2 Genetic conditions and congenital malformations affect 4-5% of all live births

Children with congenital anomalies account for 30% of admissions to paediatric wards 10% of adults have a disease with a major genetic component 5% of common cancers such as breast and colon are due to an underlying genetic predisposition Genetic testing used now to choose medication. Explain the concepts of DNA structure, repetitive DNA and polymorphisms: 1 nucleotide = base, sugar, phosphate Polymerisation of nucleotides forms nucleic acid- DNA / RNA DNA = 2 deoxribonucleic chains in double helix Complementary base pairing: Andenine-Thymine (purinepyrimidine) Guanine-cytosine Uracil replaces thymine in RNA TYPES OF DNA: Repetitive DNA is repeated in genomes but don’t code for protein: SATELITE DNA- very large, simple or moderately complex short tandemly repeated sequences, clustered round the centromeres of some chromosomes MINISATELLITE DNA-multiple copies of a 10-100bp sequence also called VNTRs (Variable Number Tandem Repeats). The original basis of DNA fingerprinting. Clustered at telomeres MICROSATELLITE DNA-single, di-, tri- or tetranucleotide base pair sequences of up to 150bp. Also known as STRs. Highly polymorphic (variable) and used in linkage studies and now DNA fingerprinting POLYMORPHISMS: Genetic polymorphisms = variation of gene in population but with NO clinical relevance. Locus Herogeneity- when faults in the same genes in same pathway lead to same condition.

• • •

Allele- a different form of a gene at a locus Genotype – an individuals genetic constitution Phenotype – the clinical outcome of an expressed gene

• • • • • •

Haploid – a half set of chromosomes Diploid – a full set of chromosomes (46) Haplotype – a particular collection of markers around an area or gene Hemizygous- when an individual has only one copy of a gene Heterozygous – when an individual has different alleles for a gene pair Heterogeneity – variation eg of an allele or locus

REPLICATION:  DNA helicase unzips double helix  Semi conservative replication therefore 1 old strand 1 new.  DNA polymerase works in 5-3 direction therefore leading and lagging strand (3-5)  In lagging stand DNA Ligase joins sugar phosphate backbone. TRANSCRIPTION:  Converts DNA into RNA  Uses RNA polymerase, 5-3, only on the non-coding strand.  Regulated by: Transcription factors binding to promoter region Enhancers and silencers TRANSLATION:  mRNA in nucleus migrates into cytoplasm  tRNA with anti codons bind to specific AAs.  Ribosomal RNA allows tRNA to line up over mRNA template so AAs can join forming polypeptide. Compare the differences between nuclear and mitochondrial genome. NUCLEAR DNA Loosly packed 1.5% coding Introns Degenerate code Mutations can effect any part of body Mutations sometimes sex linked DNA inherited = maternal + paternal If expressed gene mutated = disease

MITOCHONDRIAL DNA Tightly packed 93% coding No introns Some codons = different Aas / stop codons Mutatiopns limited to cells with high ATP need eg heart, brain, muscle Both sexes equally effected Inherited from mother Proportion of mutated Mito dictated severity codes for: 2 rRNA, 22 tRNA and

13 polypeptides

Describe chromosome structure: 1.75 coils of DNA around 8 histones forms 1 nucleosome. Further coiling of nucleosomes forms chromatin fibers. Chromatin fibers attached to non-histone proteins and after further coiling = chromosome. Centromere = point of constriction separating the short (p) and long (q) arms of the chromosome.

)-( is a metacentric chromosome, as centromere central )---(

is submetacentric, as centromere is towards the top of the chromosome Acrocentric chromosome = only stalk like structures above centromeres A telomere = tip of each end of each chromatid. Repetitive sequence important for maintaining integrity of chromosome. Compare mitosis and meiosis and discuss the importance of recombination and non-disjunction: MITOSIS Replication of somatic cells Daughter chromosome number = 2n 1 cell division mitosis = identical chromatids separated

MEIOSIS Production of gametes Daughter chromosome number = n 2 cell divisions, (can be unequal) meiosis 1--> 2 diploid daughter cells, crossing over occurs between non identical chromatids meisos 2 = identical chromatids se[parated

Unequal crossing over can result in deletions Non-disjunction = when chromosomes not separated exactly into half

GAMTEOGENESIS:

Oogenesis: Formation of oogonia, 1 mature ovum, 3 polar bodies Takes place in utero, all oogonia suspended in meiosis 1 until puberty where ~5 start to develop each month until menopause. Older mothers more likely to have non-disjunction in gametes. Spermatogenesis: Takes place from puberty onwards. 4 spermatozoa formed from 1 mother cell Older fathers more prone to mutations in gametes as more point mutations

CLS03: How the Body Responds to Infection Explain the threat posed by the many different types of infectious agents, and the requirements of an effective immune system: Infections are a major cause of morbidity and mortality worldwide An effective immune system must be able to recognise infectious agents by interacting with microbes and their components. It must also be able to defend the body against these microbes and their components. List and categorise the main cellular and secreted mediators of immunity: Cellular: White blood cells (Leucocytes) Cells in specialised tissues Cells scattered throughout most tissues of the body These cells can be categorised by their developmental origins from stem cells – myeloid and lymphoid, or by their morphology – granulocytes and mononuclear cells. Secreted: Anti-bodies Anti-toxins Regulatory substances Inflammatory substances Describe the main features of the systemic immune system, the lymphoid system and lymphocyte recirculation: Systemic immune system: Cellular components Secreted components Surface epithelial barriers Lymphocyte recirculation:

Antigens are carried from the site of infection to nearby lymphoid tissues. Some antigens are carried free in the lymph and some are captured by dendritic cells, which migrate to the lymphoid tissues carrying the antigens with them. In the lymphoid tissues, the antigens activate lymphocytes that specifically recognise them. The specifically activated lymphocytes and antibodies (which have been produced by the B-lymphocytes) can then recirculate back to the site of infection. Explain the requirements for recognition and defensive functions within the immune system: Immune recognition and defence are achieved in two ways: Components of the immune system that generate innate immunity. Components of the immune system that generate adaptive immunity.

List the main properties of innate and adaptive immunity: Innate Immunity Quickly activated when infection occurs. Remains the same on repeated exposure to the same microbe. Provides a general response to categories of microbes. Triggered by the recognition of chemical structures that are characteristic of microbes. Adaptive immunity Activated more slowly than innate. Efficacy improves with repeated exposure to the same microbe. Highly efficient and provides specific responses tailored to each individual type of microbe Outline the stages of primary and secondary immune responses, indicating the contributions of innate and adaptive components: Primary Immune Response The first infection by a particular microbe generates a primary immune response. This requires penetration of surface epithelial barriers i.e. epidermis of skin. An immediate local immune response in the infected tissues is generated by components of the immune system resident in those tissues e.g. macrophages. This also generates inflammatory mediators that attract further leucocytes and serum proteins into the infected tissues from the blood stream. This is the innate response.

While the innate response is being generated, antigens are carried from the site of infection to nearby lymphoid tissue in order to generate and adaptive immune response. Some antigens are carried free in the lymph, and others are captured by dendritic cells and carried to the lymphoid tissues. In the lymphoid tissues the antigens activate lymphocytes that specifically recognise them. These activated lymphocytes ad antibodies (produced by B lymphocytes) can then recirculate back to the site of infection. If re-infection with the same type of microbe occurs soon after the primary response, then pre-formed antibodies and effector lymphocytes will be available immediately. Secondary Immune Response If re-infection occurs later, a secondary immune response will be generated by memory lymphocytes formed during the primary response. These give a faster and bigger response than that of the primary response, hence the term adaptive immunity. Outline the different strategies of immunity required to combat different categories of infective agents: Microbes that remain outside the cell (extra-cellular) are available for being coated (opsonised) by antibodies and complement proteins, and can be engulfed and digested by phagocytes. Some of these engulfed microbes are resistant to digestion and can survive and replicate in intra-cellular vesicles and require activation signals from helper T lymphocytes to digest them. Interferon proteins can block replication of viruses in infected cells, and killer cells kill the cells themselves.

Give examples of immunopathological disorders, including immunodeficiency, allergy, transplant rejection and lymphoproliferative disorders: Immunodeficiency Disorders Particular components of the immune system are missing or defective. Primary immunodeficiencies are genetic disorders. Secondary immunodeficiencies are acquired during life e.g. AIDS. Allergies Due to inappropriate adaptive immunity against non-infective materials (allergens) that lead to inflammatory tissue damage. E.g. Allergic Rhinitis (e.g. hayfever caused by plant pollens), asthma and eczema.

Transplant Rejection Caused by antigenic differences in tissue components between donor and recipient that generate an adaptive anti-graft immune response. Lymphoproliferative Disorders Occurs where there is malignant transformation of cells of the immune system. E.g. lymphomas, leukaemias and myelomas.

CLS4: Cytogenetic Tests Recognise a normal male and female karyotype, both written and visually: Male XY Female XX Give 1 common variation seen in a normal karyotype: Somee individuals have other karyotypes with added or missing sex chromosomes: 47,XYY, 47,XXY, 47,XXX and 45,X. The karyotype 45,Y does not occur, as an embryo without an X chromosome cannot survive. Outline methods of cytogenetic analysis used in a service laboratory: “Cytogenetics is the analysis of blood or bone marrow cells that reveals the organization of chromosomes.” Molecular genetics employs several techniques to visualise different aspects of chromosomes: C-banding: Giemsa binds to constitutive heterochromatin, so it stains centromeres. R-banding is the reverse of C-banding and stains non-centromeric regions in preference to centromeres. R-bands are guanine-cytosinerich regions. G-banding is obtained by trypsin digestion followed by Giemsa stain. It yields a series of lightly and darkly stained bands. Q-banding is a fluorescent pattern obtained using quinacrine for staining. The pattern of bands is very similar to that seen in Gbanding. T-banding: visualize telomeres. FISH (Fluorescent in situ hybridization)

Be aware of the different levels of resolution obtained on analysis of blood, amniocentesis and bone marrow: Blood gives a higher resolution of G banding than amniocentesis followed by CVS (chorionic villus sampling) and finally bone marrow By changing the probes, FISH can have resolution ranging from huge chromosomes or tiny (~100 kilobase) sequences. Outline the technique of Fluorescent In Situ Hybridisation and discuss common applications: Can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. Probe DNAs are first labelled with fluorescence and then hybridized to chromosomes This addition of fluorescence can be done in various ways, e.g. PCR using tagged nucleotides. After preparation the probe is applied to the chromosome DNA and starts to hybridize. In several wash steps all unhybridized or partially hybridized probes are washed away. The sample is embedded in an antibleaching agent and observed on a fluorescence microscope. Applications examples: a) Tumour diagnosis e.g. breast cancer is associated with an abnormality on chromosome 17 b) Downs syndrome (21)  performing fish when chromosomes are not condensed (during interphase) speeds up diagnosis Explain the importance of non-disjunction in abnormalities of chromosome number: Non-disjunction is the failure of the chromosomes to properly segregate during meiotic or mitotic anaphase Results in daughter cells with abnormal numbers of chromosomes. Non-disjunctions can result in diseases or abnormalities, and occasionally aid in adaptation and speciation. List 4 common abnormalities of chromosome number and for each give brief clinical details of a clinical example: Name Downs Syndrome

Abnormality +21

Rate 15/10,000

Details 94% due to non disjunction -

Turner Syndrome

45 X

1/10,000 of female

 about 24% of cases are associated with a mosaic (mixture of normal and abnormal cells)

Edwards Syndrome

+18

3/10,000

Patau Syndrome

+13

2/10,000 of male

uncomplicated trisomy 21 Short stature, webbed neck, low posterior hairline, wide carrying angle at elbows, small nails Renal anomalies (60%), coarctation of aorta (15%), bicuspid aortic valve Growth retardation, Small mouth, clenched hands, overlapping fingers, Prominent heels Congenital heart disease (85%), renal anomalies (30%) 50% die by 2 months of age Scalp defects, narrow distance between eyes, extra digits, cleft lip and palate brain malformation (70%),congenital heart disease, renal anomalies (3060%), undescended testes 69% die by 6 months

Describe common chromosome anomalies including: translocations, duplications, deletions, marker chromosomes and be able to outline their possible implications for a patient: Deletions: A portion of the chromosome is missing or deleted. E.g. Prader-Willi Syndrome, which is caused by partial deletion of the long arm of chromosome 15 Duplications: A portion of the chromosome is duplicated, resulting in extra genetic material. Translocations: When a portion of one chromosome is transferred to another chromosome.

Marker Chromosomes: a structurally abnormal chromosome in which no part can be identified. The significance of a marker is very variable as it depends on what material is contained within the marker. In newborn babies there is a structural chromosomal risk: Unbalanced  10/10,000 births Balanced  30/10,000 births There is a carrier risk to the next generation of chromosomally unbalanced children List 4 common clinical situations in which cytogenetic analysis might be helpful: Learning difficulties/Congenital abnormalities  Cytogenetic syndromes – diagnosis/prognosis Recurrent miscarriage  Carrier of chromosome rearrangement – recurrence risk Infertility  Sex chromosome abnormality, marker chromosome, carrier of large chromosome rearrangement Prenatal diagnosis  Trisomy (eg. Down Syndrome risk), or unbalanced familial chromosome rearrangement

Interpret a simple cytogenetic result and make basic interpretations using cytogenetic terminology (ISCN): Some brief example reports straight off slides:

Amniotic Fluid – Prenatal Diagnosis:- 47,XY,+21 Abnormal male karyotype with 47 chromosomes including trisomy 21. This is consistent with Down syndrome. Mrs xxxxxxx is at increased risk of recurrence, and should be offered prenatal diagnosis in any future pregnancies. Blood – Dysmorphic baby:- 46,XX,del(4)(p16) Abnormal female karyotype with a terminal deletion of the short arm of one chromosome 4. This deletion may account for this baby’s clinical presentation. Please send blood samples from both parents to ascertain if the deletion has been inherited from a parental chromosome rearrangement or has arisen de novo.

CLS 05: Inheritance patterns and Family History Construct and draw a 3 generation family tree using correct symbols: Start with the affected person and work up, 3 generations. Require: Age Sex Affected/unaffected – if affected, at what age diagnosed Alive/dead – if dead, at what age and from what Be wary of: Adoption Half sibling

Describe the common Mendalian forms of inheritance and be able to recognise them in a family tree i.e. autosomal recessive, autosomal dominant and X-linked recessive: Autosomal dominant: Disease resulting from mutation in one allele of an autosomal gene Both sexes equally affected with frequency and severity, Children of a sufferer 50% likelihood of disease (if other parent nonsufferer) RECOGNISE- one parent of sufferer will be sufferer ...