Lab 1 Anaemia Flashcards
Haemoglobin estimation test
Drabkins method
Drabkins fluid contains potassium cyanide + potassium ferrocyanide
Haemoglobin contains ferrous ions (Fe2+) which is converted to cyanmethaemoglobin by Drabkins solution.
This is a stable red complex which can be measured spectrometry at 540nm.
What does drabkins method measure and how?
Haemoglobin concentration.
Measures amount of cyanmethaemoglobin (stable red complex) produced after mixing drabkins solution with haemoglobin and measuring at 540nm.
What is the reaction in drabkins methods
Drabkins solution contains potassium cyanide and potassium ferrocyanide which reacts with ferrous ions in haemoglobin to produce cyanmethaemoglobin (stable red complex) - measured with spectrometer at 540nm.
What is the procedure for measuring Hb concentration?
Drabkins method.
*Standard curve has to be created using Hb standard with known concentration. Make up to 2000uL (2ml)
(e.g. Hb standard = 100g/L -> Hb conc =0g/L = 0uL + 2000uL (drabkins sol.), Hb conc = 20g/L (1/5)= 400 Hb + 1600uL Drabkins solution, Hb conc = 80g/L (4/5) = 1600ul Hb + 400uL Drabkins sol.).
*Make up patient samples in microcuvettes (in duplicate).
*Incubate at room temp for 5 mins.
* Read absorbance at 540nm.
* Draw standard curve (Hb conc = x, abs = y)
* Use average of patient values to find conc on curve.
What is requirements for blood sample for Haemoglobin estimation using drabkins method
EDTA-anticoagulated blood <24hrs old.
Also need Hb reference standard to make standard curve.
How is haematocrit measured?
3/4 fill capillary tube with blood sample (EDTA-anticoagulated)
Seal end of tube and place in centrifuge with sealed end facing away from centre.
Centrifuge for 5 mins at 12000g.
Read Hct % with reader provides.
What can a MCHC over 370g/L indicate?
Blood sample may contain cold agglutinins which causes RBCs to clump together. Analyzer incorrectly reports low number of very dense RBCs but high Hb.
Corrective methods:
1. Remix EDTA tube
2. Warm blood to 37C
3. Check for autoantibodies (DAT)
4. Check slide for spherocytosis
How to differentiate between anaemia of chronic disease and IDA
ACD = Low serum iron, low TIBC, high ferritin, typically normocytic (but can be microcytic)
IDA = low serum iron, HIGH TIBC, LOW ferritin.
Microcytic hypochromic anaemia
IDA
Thalassaemia
Anaemia of chronic disease
Sideroblastic anaemia
FBC sample acceptance criteria
EDTA anticoagulated blood
<24hrs
Blood film sample acceptance
EDTA anticoagulated blood
<8hrs old.
What is reticulocyte count an indicator of?
Erythropoietic activity in bone marrow
%retic count vs ABC in anemaia patients
%retic count can be misleading due to RBCs being depleted in some anaemia leading to a falsely elevated %retic.
what does retic count help to differentiate between?
Differentiate between anaemia caused by
*low RBC production (low retic count due to BM problem)
e.g. IDA, ACD, B12 deficiency
and
*Increased RBC destruction (high retic count - bone marrow responding well)
e.g. haemolysis, acute blood loss, thalassaemia)
How many cells to count for % retic
At least 112
112 x 9 = 1000 RBCs in total
Usually 4/5 field of view
%Retic formula
reticulocytes (big square)
————————————— x 100 = __%
#RBCs (small square) x 9
(at least 112)
RBCs are ___cytic in IDA because
Microcytic bc Iron availability is decreased => haemoglobin production decreased -> new RBCs contain less Hb = smaller and paler.
Thalassaemia RBCs are ____cytic because
Microcytic = genetic defect in globin chain production leading to imbalanced Hb synthesis = RBCs contain less Hb.
RBCs are ___cytic in anaemia of chronic disease because
First normocytic and can develop to microcytic = 1. Increased hepcidin production (due to inflammatory cytokines IL-6 produced in liver) leading to iron sequestration. Hepcidin blocks ferroportin preventing iron release -> less iron reaches developing RBCs in BM leading to defective Hb synthesis.
2. Decreased erythropoiesis = inflammation suppresses EPO production reducing RBCs -> to maintain Hb concentration, developing RBCs undergo extra divisions, resulting in smaller cells.
Microcytic anaemia (4)
- IDA
- ACD
- Thalassaemias
- Sideroblastic anaemia
Normocytic anaemia (5)
- Acute blood loss
- Early IDA
- Early ACD
- Increased destruction of RBCs
- Bone marrow disorder
Macrocytic anaemia (4)
- Liver disease/alcohol injury
- Metabolic disorder (VitB12/folate deficiency)
- Bone Marrow disorder
- Megaloblastic anaemia
IDA red cell distribution width
HIGH
anisocytosis + poikilocytosis (elliptocytes, targetcells, dacrocytes)
Thalassaemia + Anaemia of CD RDW
Normal
No variation in size and shape
% reticulocyte count in anaemia patients
Misleading because it can be falsely elevated due to patients RBCs being depleted.
%retic is #retic as percentage of # RBCs.
Why is retic count HIGH in thalassaemia?
*In thalassaemia, there is genetic defect in globin chain synthesis leading to ineffective erythropoiesis
*However bone marrow is hyperactive and tries to compensate for defective RBCs by producing more reticulocytes
WHy is retic count LOW in IDA?
- in IDA there is not enough iron available for Hb synthesis so RBC production is limited.
*Cannot increase retic production due to lack of iron.
Why is retic count LOW in ACD?
- In ACD, inflammatory cytokines (IL-6) increase hepcidin which traps iron in macrophages + enterocytes, blocking its release leading to reduced RBC production.
- Inflammation also suppresses erythropoitin (EPO) reducing BM activity.
Absolute reticulocyte count formula
%reticulocyte/100 x RBCs
= ___ x10^9/L.
