Haematology Flashcards
define haematocrit or PCV
percentage of which the cells make up the total volume
40-52%
define mean cell haemoglobin
the amount of haemoglobin in an individual cell
Hb/RCC
define mean cell volume
the size of the cell
PCV/RCC
define mean cell haemoglobin concentration
the concentration of the haemoglobin in the cell
Hb/PCV
define reticulocyte count
a measure of the immature red cells
whether there are young red cells being made and active or increased red cell turnover
what are the findings in microcytic anaemia?
reduced Hb, Hct, RCC, MCH, MCHC
MCV reduced below the normal range
anaemia with small, poorly haemoglobinised red cells
blood film - small, abnormal shape, paler (less Hb), bigger area of central clearing
what is the different between latent iron deficiency and iron deficiency anaemia?
latent iron deficiency - iron stores are cleared, but cells are ok
continued iron deficiency - hypo chromic, microcytic red cells, reduction in MCV, MCH and MCHC
what are the findings in macrocytic anaemia?
reduced Hb, Hct, RCC
normal MCH and MCHC (looking at the amount of Hb in the individual cell)
MCV increased
blood film - less red cells, larger, extra lobes in the white cells (problem with cell division)
what are the causes and treatment of macrocytic anaemia?
vitamin B12 or folic acid deficiency
replacement of deficiency
do not require a blood transfusion unless severely symptomatic
describe neutrophils
2-5 lobed nucleus
Barr body - protrusion from one of the lobes and present in XX
dense nucleus
granules present from promyelocyte stage right through to the mature nucleus
contain myeloperoxidase, phosphatase, acid hydrolases
secondary granules develop from the myelocyte stage and contain collagenase, lactoferrin, lysosome
very fast turner
lifespan of 10 hours
what are the functions of neutrophils?
fighting infection
chemotaxis
phagocytosis
killing of phagocytosed bacteria
describe eosinophils
bilobed nucleus bright pink granules pale blue cytoplasm larger than neutrophils remain longer in the circulation than neutrophils
what are the functions of eosinophils?
fight infection
enter inflammatory exudates
play a role in immediate type hypersensitivity reactions and antibody-dependent parasite damage
high count in allergic reactions or parasites
describe basophils
dark granules
often overlay and obscure the nucleus
larger than neutrophils
move from the circulation into the tissues; mast cells
what are the functions of basophils?
immediate type hypersensitivity reactions
IgE attachment sites
degranulation and histamine release in allergic responses
describe monocytes
large cells bilobed nucleus pale blue cytoplasm granules in the nucleus largest white cells
what are the functions of monocytes?
killing of microorganisms
release of cytokines
rare cells in the circulation; not often seen on routine blood films
describe lymphocytes
similar size as RBC
very little cytoplasm
activated with various infections; on activation can contain large amounts of cytoplasm, sometimes with granules
describe T lymphocytes
originate in the thymus
usually parafollicular in the nodes and periarteriolar in the spleen
make up 80% of the lymphocytes in the blood
have membrane receptors for the T-cell receptor antigen
function - T helper cells, part of antibody production
describe B lymphocytes
originate in the bone marrow
have membrane receptors for immunoglobulin
function - humoral immunty
describe immunoglobulins
produced by plasma cells and B lymphocytes
part of the defence against foreign organisms
what is the treatment of infectious mononucleosis (glandular fever)?
symptomatic
occasionally require corticosteroids
must not get any ampicillin; may gets rashes with this
recovery will be quite slow
describe platelets
cellular fragments involved in clotting and bleeding
produced in the bone marrow from the megakaryocyte (contained in the cytoplasm)
what is the cause of low large platelets?
immune thrombocytopenic purpura
some reason that they are producing an antibody that is destroying their platelets
describe the pathophysiology and treatment of immune thrombocytopenic purpura
platelet turnover increased
large immature platelets released into the circulation
steroids
immunoglobulins
anti-D
splenectomy (Howell-Jolly bodies)
do not benefit greatly from platelet transfusion
not useful unless they have life-threatening bleeding
what is the cause of increased platelets?
essential thrombocythaemia
infection
reactive cause
what is the treatment of essential thrombocythaemia?
aspirin whenever the platelet count >1000
decreased the tendency to thromboembolic phenomenon
may need cytoreduction
describe platelet activation
glycoprotein IIb sticks to the injured vessel wall
this slows the platelet down to go through secondary activation; glycoprotein IIb/IIIa is upregulated/further expressed
this causes more platelets to stick together
releases 5-HT, ADP, thromboxane, fibrinogen
what are the uses of glycoprotein IIb/IIIa inhibitors?
angiography of acute MI
what drugs inhibit the substances released by platelets (causing bruising)?
SSRIs
aspirin
NSAIDs
what is the trigger for the clotting cascade?
tissue factor exposure at the vessel wall site which will activate clotting factor VII
triggers prothrombin cleavage to thrombin
what is the result of the clotting cascade?
fibrin activation with factor XIII
makes a firm clot
which factor is not essential in the clotting cascade?
factor XII
what does warfarin suppress?
protein C and S (vitamin K-dependent anticoagulants)
factor II, VII, IX, X
what is the role of antithrombin?
switches off the activation of factor XI, X, XI§
what does a normal coagulation screen indicate?
normal patient
von Willebrand’s disease (AD)
factor XIII deficiency
platelet disorders
what are the signs of bleeding disorders?
mucocutaneous (epistaxis >30mins) unexplained menorrhagia >80ml per cycle post-dental extraction post-childbirth petechiae, purpura, soft tissue haematoma loss of function
what are the causes of a deranged coagulation screen?
paracetamol overdose
liver disease
stress (shortened APTT, high factor VIII level)
tourniquet placed on the arm for a long time
traumatic venepuncture
taken from an in-dwelling line (often locked with heparin)
transport delay
heating of the bottle
high Hct (citrate acts as a diluting for clotting factors)
what are the causes of prolonged PT?
extrinsic pathway; primary factor VII problem early warfarinisation congenital factor VII deficiency early sepsis early vitamin K deficiency
what are the causes of a prolonged PT and APTT?
common pathway;
vitamin K deficiency (factor II, VII, IX, X)
oral warfarin therapy
oral dabigatran therapy
DIC; septicaemia, meningitis, malignancy (directly activate factor X)
what are the causes of prolonged APTT?
intrinsic pathway; DIC liver disease massive transfusion unfractionated heparin therapy monitoring heparin contamination in line locks oral warfarin therapy lupus anticoagulant (antiphospholipid syndrome) factor VII, IX, XI, XII deficiency
describe an APTT correction study
undertaken whenever the APTT is abnormal
if it corrects when normal plasma is added; clotting factor deficiency (ES liver disease)
if it does not correct; lupus anticoagulant present
describe D-dimer
breakdown product of clot
present in any form of inflammation
positive in >3/4s of those >60 with good health
positive; suspected case of DIC
used to see if thrombolytic therapy is working
how is D-dimer used in suspected venous thromboembolism?
negative predictive value
used with the wells score
what is the cause of bleeding with normal PT, APTT, fibrinogen?
von willebrand’s disease
factor VIII deficiency
platelet dysfunction
what is the cause of bleeding with prolonged PT only?
early warfarin deficiency
factor VI deficiency
what is the cause of bleeding with prolonged APTT only?
factor VIII, IX, XI XII deficiency
lupus anticoagulant
unfractionated heparin present (clinically or due to line contamination)
what is the cause of bleeding with PT and APTT prolonged?
oral warfarin therapy
vitamin K deficiency
oral direct thrombin inhibitor dabigatran
what is the cause of bleeding with PT and APTT prolonged and fibrinogen reduced?
hyperfibrinolysis
severe end stage liver disease
DIC
what is the problem with testing levels of protein C, S and antithrombin at the time the clot is diagnosed?
they are frequently reduced as it is the body’s response that these anticoagulants will be dissolving the clot
what are the causes of a reduced antithrombin level?
low molecular weight heparin
unfractionated heparin
what is the cause of a suppressed protein C and S level?
warfarin therapy
describe factor V Leiden
activated protein C resistance
more likely to clot
poorly penetrant; having the mutation does not guarantee that you will suffer from a VT
increased risk in pregnancy, lower limb in immobilised plaster cast
describe the prothrombin gene mutation
G20210A
very weak increase in the risk of a deep vein thrombosis but not in a PE
describe antiphospholipid syndrome
autoimmune
hyper coagulable state
promotion of blood clots in the arteries and veins
positive lupus anticoagulant
what blood disorders can be caused by anti-D antibodies?
haemolytic disease of the new-born
transfusion reactions
define agglutination
the aggregation of antigens and antibodies
haemagglutination in red cells
describe the expression of A antigens
A1 - vey strong expression
A2 - very weak expression
describe reverse grouping
testing patient plasma for anti-A and anti-B
patient plasma is added to suspensions of group A1 and B red cells
haemagglutination; B + anti-B; blood group A
describe antibody screening
used to screen patient plasma for clinically significant red cell antibodies other than anti-A and anti-B
haemagglutination; red cell antibody present
describe forward grouping
testing for the presence of A and B antigens on patient red cells
suspension of patient red cells is prepared, monoclonal antiserum and anti-A or anti-B is added
haemagglutination; anti-A + A antigens; blood group A
describe an antibody identification test
performed if the antibody screening test is positive
if a red cell antibody is identified, then red cells lacking that antigen must be selected for crossmatch
describe the electronic crossmatch
faster response to requests
less wastage
reduction in laboratory workload
cannot be used for patients with clinically significant red cell alloantibodies
describe serological crossmatch
the addition of patient plasma to a suspension of donor red cells
what feature are essential for an intact and functioning haemoglobin molecule?
2 properly formed alpha and beta globin chains each
iron
haem molecule
describe the beta globin genes
on chromosome 11
4 genes; epsilon, gamma, delta, beta
describe the alpha globin genes
on chromosome 16
4 genes; 2 alpha, 2 zeta
name the haemoglobin that occur in humans
HbA; alpha 2 beta 2 HbA2; alpha 2 delta 2 HbF; foetal, alpha 2 gamma 2 Hb portland; embryo, zeta 2 gamma 2 Hb Gower 1; embryo, zeta 2 epsilon 2 Hb Gower 2; embryo, alpha 2 epsilon 2
define haemoglobinopathies
serious anaemias caused by the inheritance from both parents of changes in the structure/synthesis of the individual globin chains of haemoglobin
usually inherited in AR
parents are usually healthy carriers
describe the pathology of haemoglobin E, C, D
single point mutations in an individual globin gene
why have haemoglobinopathies become so widespread?
protection from malaria
describe alpha thalassaemia carrier state
genetic defect in the production of one or more of the alpha-globin genes
symptomless carrier state
common in Southeast Asia or eastern mediterranean
describe the disease of alpha thalassaemia
severe disease; haemoglobin bart’s, hydros foetalis
no production of alpha-globing correctly
death of the baby in utero or soon after birth; severe anaemia
serious clinical problems for the mother during pregnancy
describe the pathology of alpha thalassaemia
Hb bart's in foetus; 4 gamma globin genes Hb H in adults; 4 beta globin genes high oxygen affinity severe tissue hypoxia inclusion bodies membrane damage red cells are broken down by haemolysis compensatory hypersplenism; unsuccessful severely anaemia foetus/baby
what are the symptoms and signs of a pregnancy woman with alpha thalassaemia
mild anaemia low Hb low MCV normal WCC, PLT, MCH, MCHC blood film; mild, minor changes in red cells
describe the inheritance of alpha thalassaemia
3/4; compatible with life, some normal adult haemoglobin made, will grow to adulthood, quite anaemic
4/4; born with severe anaemia, probably die shortly after birth, alpha thalassaemia major
what are the signs and symptoms of alpha thalassaemia major?
blood film;
severely abnormal red cells; large, pale, containing very little Hb
nucleated RBCs; increased red cell turnover, normally not seen in a neonate
splenomegaly
severely anaemic
in heart failure
describe the carrier state of beta thalassaemia
defect in the beta-globing gene
failure of production of the beta-globing protein
symptomless carrier state
common in the mediterranean, Middle East, Indian subcontinent, south east Asia, pacific island populations
what are the symptoms and signs of beta thalassaemia major?
low Hb normal WCC, PLT anaemic from birth film; poorly haemoglobinised red cells, with many abnormalities, nucleated RBCs small for their age prominent head soft feeling to the head; bone marrow cavity is expanded to make increased amount of blood to compensate for haemolysis and failure of normal Hb production hepatic splenomegaly
why is beta thalassaemia major compatible with life?
first 6 months of life there is HbS; foetal Hb, containing 2 alpha and 2 gamma chains
only as the beta globin chains take over as the normal adult Hb they become increasingly anaemic
failure of production of adult Hb
what is the treatment of beta thalassaemia major?
transfusions
try and dampen down their own haematopoiesis as much as possible
entirely dependent on a transfusion regime
what are the symptoms and signs, if any, of beta thalassaemia carrier state?
mildly anaemic
microcytic picture
normal MCHC
blood film; quite a lot of red cell abnormalities, variation of size and shape, pale, poorly haemoglobinised red cells
describe the thalassaemia trait indices
similar in both alpha and beta globin defects low Hb microcytosis normal/raised RCC low MCH normal MCHC
what are the symptoms, signs and maintenance of thalassaemia trait?
microcytic anaemia due to globin gene defect
normal RCC and MCHC
require ferritin measurement to prove if they are iron deficient
require genetic screening
have some protection against malaria
define sickle cell disease
caused by a single mutation in the beta globin gene at position 6, leading to an amino acid change of glutamic acid to valine
describe sickle cell carrier state
common in tropical Africa, the Middle East, India, caribbean islands, south america
AR inheritance
describe the signs and symptoms of sickle cell disease
low Hb
normal WCC and RCC
blood film; red narrowed (sickle) cell
what are the causes of cell shape changes in sickle cell disease?
lack of oxygen
are later destroyed or haemolysed
destruction; leads to many of the problems in the disorder
irreversible change
what are the clinical features of sickle cell anaemia/haemolytic anaemias?
chronic red cell breakdown; jaundice, yellow sclera
splenomegaly
bony lesions; pain, crises
severe pain with trauma, infection, cold exposure, dehydration
chronic ill health
repeated cerebral infarctions; strokes, major long-term morbidity
what is the management of sickle cell anaemia/haemolytic anaemias?
treatment the complications and symptoms transfusions screening; minus Northern European neonates; heel prick test antenatal; ideally pre-conception, anybody at risk of HbS, alpha or beta thalassaemia
what does morphology refer to?
appearance of the cells
in blood, bone marrow, lymph node or other tissues
describe the morphology of acute promyelocytic leukaemia
larger number of large cells
abundant cytoplasm
nucleus almost obscured by the presence of large granules within the cytoplasm
describe the morphology of acute lymphoblastic leukaemia B-lineage
pleomorphic population of large cells
convoluted nuclei
prominent nucleoli
clearly blast cells, but cannot be certain of the lineage; myeloid, B or T lymphoid
describe the morphology of hairy cell/chronic B lymphocytic leukaemia
oval/round nucleus
cytoplasm with abundant villous projections
similar cells are found in other disorders
describe the immune-phenotype of a cell population
the pattern of antigen expression
on the cell surface and intracellularly
assessed by flow cytometry
describe flow cytometry
cells in suspension pass in single file through a laser beam
the cell momentarily breaks the beam and scatters the light
the light is collected by a combination of filters, mirrors and detectors and the data is recorded
what cell suspensions are measured by flow cytometry?
blood
bone marrow
CSF
pleural fluid
how are cell population identified in flow cytometry?
physical characteristics, scatter
antigen expression, fluorescence
how is antigen expression assessed in flow cytometry?
cells are incubated with an antibody linked to a fluorochrome
the number of cells and intensity of antigen expression depends on the strength of the light emitted
what does an antibody panel assess?
cell lineage; myeloid or lymphoid origin, B, T or NK cell
stage of differentiation; degree of maturity
describe CD45
the common leukocyte antigen
expressed on all white blood cells
what is the pattern of antigen expression on AML?
CD34 CD33 CD13 myeloperoxidase HLA-DR CD117
what is the immune-phenotype of acute lymphoblastic leukaemia?
weeks CD45 expression
lower side scatter
positive for TDT, CD10, CD19, HLA-DR
describe cytogenetics
refers to the chromosomes of the cell population
cell division is arrested at metaphase
giemsa staining produces specific bounding patterns along the chromatids
examined under a high power microscope
identify individual chromosomes and produce a karyotype
describe the abnormalities of a karyotype in haematological malignancies
numerical abnormalities; extra or missing copies
structural abnormalities; translocations, inversions, deletions
diagnostic; CML, APL
prognostic; acute leukaemias
describe FISH
a single standard DNA probe anneal to its complementary sequence in the target genome
detects and localises specific DNA sequences
what are the advantages of FISH?
can visualise abnormalities in non-dividing cells; useful in CLL with low mitotic index
can be performed on directly-prepared samples rather than requiring cell culture
larger number of cells can be analysed
can detect cryptic rearrangements
can be performed on tissue sections; bone marrow trephine biopsies
describe FISH probes
chromosome paints; hybridise to metaphases to visualise different chromosomes at the same time, can determine the origin of structural abnormalities
locus-specific probes; target genes of interest, identify rearrangement, deletions or gains in metaphase and interphase
describe the diagnosis of acute promyelocytic leukaemia
must be confirmed by cytogenetic analysis
defining chromosomal abnormality; translocation between long arm of chr 15 and 17; PML-RARA fusion gene
cryptic abnormalities can result in a PML-RARA fusion gene; detected by a FISH probe for RARA gene on chr 17
describe the diagnosis of AML
abnormalities of chr 5, 11, 15, 17 and a complete loss of chr 8
specific loss of the long arm of chr 5; poor prognosis
define PCR
a method by which DNA is amplified to produce thousands of copies of the same sequence
define taq polymerase
the thermostable/heat stable enzyme which extends the primers by the sequential addition of nucleotides to synthesise a complementary DNA strand from the original template
define deoxynucleoside triphosphates/dNTPS
the building blocks used by Taq polymerase to synthesis the new DNA strand
describe the process of PCR
denaturation at 95 degrees; separation of the double strands of DNA
annealing; cooling of the reaction
primers bind to their complementary sequences
higher temperature; taq polymerase carries out the extension phase
dNTPs are added to synthesise the new strand
describe gel electrophoresis
analysing PCR product
migration through an agarose gel
separation of the reaction products by their size alongside a molecular weight marker
visualised using ethidium bromide (EB) staining and UV light
describe reverse transcriptase PCR
converts messenger RNA to complementary DNA (or cDNA)
describe allele-specific PCR
uses primers which include a known mutation
detect the JAK2 V617F mutation; myeloproliferative neoplasm
what are the clinical applications of PCR?
BCR-ABL fusion gene detection; CLL philadelphia positive variant; ALL JAK2 V617F and MPL mutations; myeloproliferative neoplasms FLT3 and NPM mutation; AML PML-RARA; acute promyelocytic leukaemia
describe the morphological features of CML
blood; neutrophil leukocytosis granulocyte prescursors at all stages of maturation basophilia prominent degree of eosinophilia blasts present, not increased high platelet count
bone marrow aspirate; markedly hypercellular granulocytic hyperplasia normal differentiation basophils and eosinophils prominent blasts present, not increased megakaryocytes; smaller, reduced nuclear lobulation
describe the cytogenetic findings of CML
reciprocal translocation between the long arms of chr 9 and 22
abnormal chr 22; philadelphia chromosome
BCR-ABL1 fusion oncogene
abnormal protein function directly produces the clinical and haematological phenotype of CML
loss of Y chromosome
what is the management of CML?
TKI; imatinib 400mg daily
monitor haematological response by blood counts or cytogenetic analysis
BCR-ABL1 transcript level; measured by real-time PCR
complete molecular response; BCR-ABL1 fusion gene is no longer detectable
rise in transcript levels; therapy failure, development of mutations within BCR-ABL fusion gene
describe the BCR-ABL dual fusion FISH probe
normal red signal, normal green signal and 2 abnormal fusion signals (yellow)
describe the symptoms and signs of CLL
weight loss sweats malaise widespread lymphadenopathy hepatosplenomegaly raised WCC mild anaemia thrombocytopenia
describe the symptoms and signs of CML
fatigue
left hypochondriac discomfort; splenomegaly
raised WCC
moderate thrombocytosis
describe the morphology of CLL
blood;
small cells
very scant cytoplasm
condensed nuclear chromatin
bone marrow trephine;
nodular infiltrate of small cells in the peripheral blood
describe the flow cytometry of CLL
positive for CD19, 20, 5
negative for CD10
weak expression of surface light chain
what features indicate advanced CLL?
anaemia thrombocytopenia lymphocyte doubling time CD38 expression on lymphocyte surface 17p deletion presence; more aggressive and failure to respond to purine analogue therapy
what is the treatment of CLL?
absence of 17p deletion; rituximab, fludarabine, cyclophosphamide
