testing haematology - anaemia

when doing a blood cell count what anticoagulant is used

EDTA - salt, doesnt dilute the blood

what does a full blood count analyse 5

• haemoglobin level

• white blood cell count - looks at levels of dif types of wbc (automated differential)

• red blood cell count

• platelet count

• red cell parameters

centrifugation of anticoagulanted blood

normal haemoglobin values

MEN    13 - 18 g/dl 

WOMEN  12 - 15 g/dl

Red Blood Count (RBC)  4.5-6.5x10¹²/l  3.8-5.8x10¹²/l

Mean Cell Volume (MCV)  78-93 fl

Mean Cell Haemoglobin (MCH)  27-32 pg

Packed Cell Volume (PCV)  0.40-0.52 l/l  0.37-0.47 l/l

Reticulocyte Count 0.2-2.0%

normal wbc value

5 – 10 x 10⁹/l

Neutrophils  2.0-7.5 x 10⁹/l  40 - 75%

Lymphocytes  1.5 – 4.0 x10⁹/l  20 – 45%

Monocytes  0.2 – 0.8 x10⁹/l  2 – 10%

Eosinophilia  0.04 – 0.4 x10⁹/l  1 – 6%

Basophils  <0.01 – 0.1 x10⁹/l  1%

normal platelet values

150 – 400 10⁹/l  

how haemoglobins measured

►Measured by haemiglobincyanide (HiCN) method

►Blood diluted in solution containing potassium cyanide and potassium ferricyanide

►Hb, Hi, and HbCO converted to HiCN.

►The absorbance of the solution is measured in a spectrophotometer at 540nm

►Can use Sodium lauryl sulphate instead of KCN to reduce toxicity.

mean cell volume =

Hct (haemltocrit) x 1000/rbc

size of RBC

►MCH - mean cell haemoglobin

►MCH = Hbx10/RBC

haemoglobin count

►MCHC - Mean cellular haemoglobin concentration

►MCHC = Hb/Hct

RDW - Red cell distribution width

►This is a volume distribution histogram which allows different populations of red cells to be seen.

►Expressed as a CV of the red cell volume (%)

►Can all be used as diagnostic markers

 Reticulocyte Counting

►RBCs released from the Bone marrow in the last 3 days

still has Ribosomal RNA

(normal rbc dont have nucleus)

Romanowski Stain - pH 6.8

methylene blue - stains nucleus plus

eosin y - stains haemoglobin

Normal Peripheral White Blood Cells

what is anaemia

►A reduction in oxygen-carrying capacity due to a lower haemoglobin concentration than is usual for that individual.”

►Anaemia occurs when red cell destruction is greater than red cell production.

Classification of anaemia

morphologic classification based on appearance and size of rbc

►Red cell indices obtained from analysers.

§Haematocrit

§ Mean cell volume

§Mean cell haemoglobin

red cell morphology

haemoglobin structure

§Four globin chains (2 alpha, 2 beta).

§Each globin chain has an iron containing haem molecule.

The iron in thehaemmolecule binds to oxygen

genetics

• in foetus - Hb F

• ►In normal adults 96 – 98% of haemoglobin is HbA,  Hb A2 (2 – 3%) and HbF (<1%) constitute a minor component of the total haemoglobin.

geography

thalassaemia

§is a reduced rate of synthesis of globinchains.

Defined by imbalance αβ ratio

• lower rbc count and haemoglobin count

alpha thalassaemia classification

• gene deletion

• ►α trait due to deletion of one or two of the four alpha genes, asymptomatic              (eg - α/ α α, --/ α α, - α/- α).

  ►Haemoglobin H disease is the lack of three of the four α genes resulting in alpha thalassaemia major.

  ►Haemoglobin Bart’s Hydrops Foetalisresults from absence of all four αgenes, incompatible with post natal life.

beta thalassemia classification

• reduction in beta chain production

• β thal major - severe phenotype

• β thal minor - asymptomatic

• β thal intermedia - intermediate phenotype

• ►β 0 syndromes are characterized by the affected gene producing no beta chain.

  ►β + syndromes are characterized by the abnormal gene producing beta chains at a reduced rate.

  ►Usually due to point mutations.

variant haemoglobin classification

disorders of globin chain synthesis

Clinical features of b - Thalassaemia major

• iron overload causes liver damage ]

• delayed or absent puberty

• Diabetes

• excess melanin

• prone to infection

b - Thalassaemia major treatments

►Regular blood transfusions

►Regular folic acid.

►Iron chelation therapy.

►Vitamin C

►Endocrine therapy

►Immunisation against Hep B

►Bone marrow transplantation.

Automated Cell Counting methods
(Erythrocytes / Leucocytes / Platelets) 2

• electricalimpendance

• laser light scatter

electrical impedance

the electrical resistance of a particle passing between 2 electrodes is proportional to the particles volume

laser light scatter

the light scattered by a particle passing a beam of laser light is proportional to the particles volume

haemoglobin abalysis - spectrophotometric methodology

Absorbance proportional to Hgbconcentration

►

  (Hgb less than optimum = Anaemia)

fbc thalassaemia

►RBC often increased in thal but decreased in iron deficiency and ACD.

►Hb typically normal in thal minor but decreased in intermedia and major syndromes.

►MCV is the most valuable parameter in predicting thal.

Hb H inclusions

►Hb H is an insoluble tetramer consisting of four beta globin chains, due to a lack of alpha chains in alpha thal major.

►In Hb H disease 30 – 100% of RBCS contain Hb H inclusions.

►In alpha thal minor there is one cell with Hb H inclusions per 1000 – 10,000 RBCS

►When there is a reticulocytosis a rare Hb H inclusion may be missed – operator experience crucial.

►Detection of Hb H inclusions points to an alpha chain mutation and narrows the amount of DNA analysis required.

►False negatives problematic, even with 2 alpha gene deletions no inclusions may be seen after several minutes of searching.

Electrophoresis principles

►Separation of haemoglobins with electrophoresis at pH 8.4 (alkaline) and pH 6.2 (acid).

►Scanning allows quantification of the haemoglobin present, bands are seen by staining.

►At alkaline pH Hb C, E, A2 and O migrate together to form a single band, Hb S, D and G also co migrate.

Electrophoresis interpretation

HbA2 range	Interpretation
> 7.0 %	Rare, repeat to verify test. Exclude a structural variant. Can be due to rare β thal mutations.
3.8 – 7.0 %	Beta thal trait  or unstable Haemoglobin.
3.4 – 3.7 %	Fe deficiency in β thal trait; Δ chain variant with β thal trait. Interaction of α and β thal traits; rare β thal mutations. HbS making measurement inaccurate; interaction of α - Hb S.
2.0 – 3.3 %	Normal. Δ and β thal (but HbF should be elevated); alpha thal trait. Rare cases of β thal trait coexisting with either Δ or α thal trait.
< 2.0 %	Δ β thal (but HbF should be elevated). Alpha thal trait; Hb H disease; Δ variant or delta Thalassemia. Iron deficiency.

sickle solubility tests

►Detects HbS at conc. > 20% and differentiates HbD and G which migrate with Hb S on cellulose acetate electrophoresis at alkaline pH.

►Positive results are also obtained on samples containing both HbS and beta globulin mutations.

►False positives can occur in leukocytosis, hyperprotienemia and unstable hemoglobin states.

►False negatives can occur in patients with anaemia or if outdated buffer is used and in infants less than 6 months.