methods in haematology II Flashcards
Mean Cell Haemoglobin (MCH) =
Haemoglobin (Hb) divided by Red cell count (RBC)
Mean Cell Volume (MCV) =
Packed cell volume(PCV) divided by Red cell count (RBC)
Mean Cell Haemoglobin
Concentration (MCHC) =
Haemoglobin (Hb)divided by Packed cell volume(PCV)
Haematocrit (HCT or PCV packed cell volume)
Can be calculated based on red cell count & size OR measured in centrifuged capillary tube.
- Males 40-52%
- Females 38-48%
what is haemoglobin typically measured in
Haemoglobin is typically measured in aggregate on lysed cells and compared to the red cell number and size]
what is red cell number and volume measured by
Red cell number and volume is measured by scatter or impedance
- Mean corpuscular haemoglobin (MCH)
- Mean corpuscular haemoglobin (MCH) is a calculation of the amount of oxygen-carrying haemoglobin inside your RBCs.
Mean corpuscular volume (MCV)
Mean corpuscular volume (MCV) femtolitres calculated from PCV/RBC count or averaging of red cell size
would macrocytic RBC have higher MCH (mean corpuscular haemoglobin)
Since macrocytic RBCs are larger than either normal or microcytic RBCs, they would also tend to have higher MCH values.
- Mean corpuscular haemoglobin concentration (MCHC)
is a calculation of the concentration of haemoglobin inside the RBCs.
what is decreased MCHC (mean corpuscular haemoglobin concentration) seen in what conditions
Decreased MCHC values (hypochromia) are seen in conditions where the haemoglobin is abnormally diluted inside the red blood cell
cells, such as in iron deficiency anaemia, long standing inflammation or thalassaemia.
increased MCHC mean corpuscular haemogolbin concentration diseases
are seen in conditions where the haemoglobin is abnormally concentrated inside the red cells, such as in hereditary or
autoimmune spherocytosis.
- Red cell distribution width (RDW)
) is a calculation of the variation in the size of your RBCs (by impedance or cytometry).
RDW-SD is the standard deviation, RDW-CV is the coefficient of variation (SD/Mean).
Anti inflammatory tests
c-reactive protein – good indicator up to 24h in inflammatory response
plasma viscosity and ESR- for monitoring
blood analyser flow cytometry =
sample typically separated to measrure RBC/ platelets and WBC.
the blood smear
Geimsa stain - Methylene blue, Azure B & Eosin
(Wright stain - Methylene blue & Eosin)
Erythrocytes - pink,
Platelets - light pale pink
Lymphocyte cytoplasm - sky blue
Monocyte cytoplasm - pale blue
Leukocyte nuclear chromatin - magenta
Eosinophil granules - red
Basophil granules - blue/purple
ELIZA enzyme linked immunosrbent assay
1) Coated antibody specific for protein masured
2) Plate washed remove excess
3) Plasma is added binds to the immoblised antibody
4) Plate is washed removes plasma and protien
5) Addition of antibody that is conjugated to an enzyme the antibody binds to the protien.
6) Plate is wajsed and rmoved excuesses antioby and fluid
7) Substarte is reacted with the enzyme to produce a colour change.
Assays for clotting disorders
- Prothrombin time (PT): measures extrinsic
pathway – measures clot formation upon the
addition of thromboplastin - Thrombin Time (TT): measures clot formation
upon addition of thrombin - Fibrinogen: measures adequate levels of
fibrinogen for clot formation
LIA
Latex immunoassay (LIA)
Latex particles coated with antibody of protien being measured , agglutation measured turbidmetrically
Activated partial thromboplastin (APTT) measured the intrinsic pathway
which measures clot formation in the presence of APTT reagent and calcium ions
micronutrients (vitamins /Minerals) required for RBC production
iron , vitamin B12 , folate , Vitamin B6 , ferritin
the uptake of iron
iron is absorbed from the duodenum and jejnum
best absorbed is the Fe2+ form , is it transported in plasma bound to transferrin which delivers itr to tissues these tissues have transferrin receptors, it is stored in tissues bound to ferritin.
what is Hepcidin
(peptide hormone produced by liver) regulates iron uptake from gut by blocking iron export from cell by ferroportin and degrading ferroprotein
what is required for DNA synthesis
Vitamin B 12 and folate
Red blood cell composition ?
alpha chain , beta chain , heme group beta chain , alpha chain 2 (helical shape of the polypeptide molecule.
what is Anaemia
Low red cell number or low levels of Hb
<13g/dL (men), <12g/dL (women), <11gdL (pregnant women)
Usually a local reference range:
13.3-16.7g/dL men
11.8-14.8g/dL women
Abnormal and malignant haematopiesis
haemophilla and clotting disorders VWF and clotting cascade , CML, AML, MPD pathogenesis , MM ALL , CLL pathogens
Stem cells
Can self renew
Few in number
Can differentiate into several lineages
One stem cell can produce 1x106 cells after 20 divisions
Cannot be distinguished morphologically – requires functional assays
Usually located in a protected environment ‘tucked away deep in tissues’
away from immediate harm
Hypoxic niches, frequently glycolysis rather than oxidative phosphorylation
Haematopoietic stem cells
CD34+ &/or CD133+
(cluster of differentiation marker)
c-kit high
Sca-1 high
Lin- lineage negative
i.e. defined by what they don’t express
Lin- cocktail different markers
CD3: A marker for T lymphocytes.
CD4: A marker for helper T cells.
CD8: A marker for cytotoxic T cells.
CD19: A marker for B lymphocytes.
CD20: Another marker for B lymphocytes.
CD14: A marker for monocytes.
CD16: A marker for natural killer (NK) cells & some
monocytes.
CD56: A marker for NK cells
Osteoblasts:
factors maintain HSC quiescence
(a non-dividing state) and support their self-
renewal.
Endothelial Cells:
factors that promote HSC
proliferation and differentiation.
Mesenchymal Stem Cells (MSCs):
source of
stromal cells and secrete regulatory molecules
that influence HSC behavior. They become
osteoblasts (bone-forming cells),
chondrocytes (cartilage cells), and adipocytes
(fat cells).
Macrophages:
IL-6 and TNF-α can promote HSC proliferation, IL-10 can induce quiescence
Stem cells:
Pluripotent
can renew or
differentiate into all
haematopoietic
lineages
Multipotent – can
differentiate into
multiple lineages
Lineage committed
can only develop
along that ‘line’ of
differentiation
Fully differentiated
cells with function in
blood
haematopoeisis is required for
transpprt of oxygen
fight infection
haemostasis (blood clotting)
transport of nutrients and removal of toxic products
cellular processes invovled
ØProliferation – cell division and growth
ØDifferentiation – acquisition of specific maturation characteristics for function
ØApoptosis - cell death
* Healthy individuals: 5-10 x 1011 blood cells per day
Where haemtoposiesis takes place ?
Sternum , pelvis , femurs
Normal bone marrow
- Haematopoietic tissue: stem
cells and progeny - Mesenchymal stem cells that
produce stromal cells and
fibroblasts that secrete
scaffolding proteins such as
collagen - Macrophages: produce growth
factors to regulate
haematopoiesis - Adipocytes: store energy in form
of fat
Pluripotent: able to produce progeny
of all lineages
Oligopotent: able to produce
progenitor cells that may differentiate
into a few cell types ie lymphoid or
myeloid stem cells
Myeloblast –
myeloid pathway
Lymphoblast –
lymphoid pathway
Erythroblast –
erythrocyte pathway
Colony Forming Assays
Diagnose leukemia, myelodysplastic syndromes (MDS), aplastic anaemia
& monitoring of chemotherapy and transplantation
Colony-forming unit–granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-
GEMM)
Colony-forming unit–lymphocyte (CFU-L)
Colony-forming unit–erythrocyte (CFU-E)
Colony-forming unit–granulocyte-macrophage (CFU-GM)
Colony-forming unit–megakaryocyte (CFU-Meg)
Colony-forming unit–basophil (CFU-B)
Colony-forming unit–eosinophil (CFU-Eos)
growth factors and cytokines
Interleukins (IL) SCF (IL-11) – proliferation and development of
pluripotent and progenitor cell development
IL-1 stimulates production of GM-CSF, G-CSF, M-CSF
and IL-6
IL-3, IL-4 and IL-6 act on early multipotential cells
IL-5 is eosinophil CSF
Colony Stimulating Factors
(CSF)
G-CSF stimulates differentiation of granulocyte
precursors and activates mature granulocytes
GM-CSF stimulates differentiation of granulocyte and
macrophages
Other growth factors Erythropoietin (EPO) – released from kidney and
stimulates red blood cell production in bone marrow
Thrombopoietin (TPO) – produced by liver and
stimulates megakaryocyte maturation and platelet
production in bone marrow
IL-1
stimulates production of GM-CSF, G-CSF, M-CSF
and IL-6
IL-3, IL-4 and IL-6
act on early multipotential cells
IL-5
is eosinophil CSF
G-CSF
stimulates differentiation of granulocyte precursors and activates mature granulocytes
GM-CSF
stimulates differentiation of granulocyte and
macrophages
Other growth factors Erythropoietin (EPO)
released from kidney and
stimulates red blood cell production in bone marrow
Thrombopoietin (TPO)
produced by liver and
stimulates megakaryocyte maturation and platelet
production in bone marrow
Erythropoiesis
Life span of RBC is 100-120 days
20 seconds to circulate
2-3 x 1013 RBC normally
¼ of the body’s cells
Granulopoiesis
Granulocytes: Neutrophil (3-
5 lobes), Basophil (2 lobes),
Eosinophil (2 lobes).
Distinct subset of granules
containing Bioactive
enzymes for function
Thrombopoiesis
(platelet production)
* Platelets for blood clotting arise from the
megakaryocyte (Mk) via the CFU-EMk
progenitor
* Mk’s are polyploidy – have multiple sets
of chromosomes (up to 64N as opposed
to 2N diploid)
* Thrombopoietin (TPO) essential for Mk
growth and development of platelets and
platelet function – agregration process
Monocytes
- Arise from the Granulocyte Monocyte lineage CFU-GMo
- Monocytes pass into circulation then migrate into tissues and differentiate into
macrophages, they can then further differentiate into more specialised cells
such as Langerhans cells etc - Act as immune-surveillance systems
Lymphopoiesis
T cell development
CLP’s in BM produce double
negative (DN) cells (CD4 and CD8
negative) migrate to thymus,
development allows expression of
double positive cells (CD4+CD8+)
then generating MHC class I
restricted CD8+ cytotoxic T cells
and helper MHC class II helper T
cells. T regs express CD4+ CD25+
B cell development
CLP’s generate immature B cells
which migrate to lymphoid tissues
to encounter antigen. Activated B
cells secrete IgM or IgG antibodies
or may differentiate into plasma
cells
