HB Estimation

Hb investigation is done to investigate haematological disorders that include anaemia, leukemia

and polycythaemia.
Hemoglobin content of whole blood is determined by photometric methods and recorded as
gm/dL. (Normal Ranges: males 16.0 ± 2.0; females 14.0 ± 2.0).
Methods of HB estimations
1) Visual - colour comparison is done using naked eye. Test include ;- Acid haeamatin , and
spencer.
2) Colorimetric Method- colour comparision is done using a colorimeter. Tests include ;-
Alkaline haematin, cyanmethaemoglobin, oxyhaemoglobin
3) Electronic values are determined using electronic counters eg coulter counter .
4) Spectrophotometric methods
Sahli or Acid Haematin Method
Principle - Haemoglobin (Hb) is converted to acid haematin by addition of 0.1 N Hydrochloric
acid and the resulting brown colour is compared with standard brown glass reference blocks.
Cyanmethaemoglobin Method.
Principle - Hb, Methaemoglobin, Carboxyhaemoglobin but not sulphaemoglobin are converted
to cyanmethaemoglobin when blood is diluted in a solution containing potassium cyanide and
potassium ferricyanide. The absorbance of the solution is then measured in a photoelectric
calorimeter at a wavelength of 540 mm or with a yellow-green filter.
Method
 0.20ul of blood are added to 5 ml of Drabkins cyanide-ferricyanide solution and mixed
well.
 After 10 mins, the absorbance of the solution is read against a reagent blank (or distilled
water) at 540mm.
 A standard curve or a standard table is prepared using cyanmethaemoglobin graded
dilutions of a referance solution of a known concentration and the same calorimeter.
Abnormal plasma proteins or a high leukocyte count may give erronously high Hb content due to
turbidity. The latter can be avoided by centrifuging the diluted sample. Abnormal plasma
proteins can be solubilised by addition of a detergent eg.Nonidet P40.
Alkaline Haematin Method
Haemoglobin, methaemoglobin, carboxyhaemoglobin and sulphaemoglobin are converted to
alkaline haematin by addition of sodium hydroxide - a strong alkali. It forms a true solution and
the brown colour can be read against comparable standards or in a colorimeter. Foetal
haemoglobin and Hb-Barts are alkali-resistant, but can be converted by heating in a boiling water
bath for 4 min. or by collecting the blood first in to acid and then adding alkali(acid alkali
method).
Haldanes Carboxyhaemoglobin Method
Haemoglobin is converted to carboxyhaemoglobin (which is bright red in colour), by exposing it
to carbon monoxide. It is a relatively accurate method but carbon monoxide is dangerous.
Oxyhaemoglobin Method
Haemoglobin is converted into oxyhaemoglobin by mixing blood with a dilute solution of
sodium carbonate or ammonium hydroxide. The intensity of the colour obtained is measured
colorimetrically. It is a fast and accurate method, but traces of copper can give errors.
The normal ranges are:

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  Newborns: 17 - 22 gm/dl
 One week: 15 - 20 gm/dl
 One month: 11 - 15 gm/dl
 Children: 11 - 13 gm/dl
 Adult males: 14 - 18 gm/dl
 Adult females: 12 - 16 gm/dl
 Men after middle age: 12.4 - 14.9 gm/dl
Women after middle age: 11.7 -m 13.8 gm/dl
Red Cell Count (RBC)
This measurement is made with a microscope and a specially ruled chamber (hemacytometer).
The RBC is recorded as millions of cells per cubic millimeter (Normal Ranges: males, 5.4 ± 0.8;
females, 4.8 ± 0.6).
PCV Packed Red Cell Volume (Hematocrit, Hct., PCV)

Hematocrit is the percent volume of whole blood occupied by red blood cells and is determined
by centrifuging blood in special (hematocrit) capillary tubes. The percent of whole blood which
is made up of red cells is determined (Normal Ranges: males 45.0 ± 5.0; females 42.0 ± 2.0).
RBC, Hgb & Hct From Automated Analyser Example
The RBC = 3.5 x 1012/L
Haemoglobin =10.5 g/dL
Haematocrit = 31.5%
Rule of Three
RBC X 3 = Hgb
3.5 X 3 = 10.5 g/dL
Hgb X 3 = Hct ± 3%
10.5 X 3 = 31.5 %

The Erythrocyte or RBC indices

They calculations that are made from the three main red blood cell test,
 RBC count,
 haemoglobin
 hematocrit tests.
The erythrocyte indices include the mean corpuscular volume (MCV), mean corpuscular
hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).
The calculations:-
MCV = hematocrit ÷ rbc count
MCH = hemoglobin ÷ rbc count
MCHC = hemoglobin ÷ hematocrit
The erythrocyte indices reflect the size and hemoglobin content of individual red blood cells.
Abnormal erythrocyte indices are helpful in quickly narrowing down the potential causes, and
thus classifying, the different types of anaemia. This allows the medical practitioner to prescribe
the correct treatment.
How anaemias are classified using the erythrocyte indices:-
1. MCV, MCH and MCHC are all normal - normocytic, normochromic anaemia (RBC's size
and hemoglobin content are within normal limits), most often caused by acute blood loss.

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2. MCV, MCH, and MCHC are all decreased - microcytic, hypochromic anaemia (RBC's are
smaller and their hemoglobin content is much less) - most often caused by iron deficiency but
sometimes by impaired production of hemoglobin.
3. Increased MCV with variable MCH and MCHC - macrocytic anaemia (RBC's are larger ) -
most often caused by Vitamin B12 deficiency (due to pernicious anaemia) and folic acid
deficiency.
Mean corpuscular volume (MCV)
This is probably the most important of the erythrocyte indices.
It calculates the average size of individual red blood cells and thus categorises them, eg:-
Normal size cells are called normocytic
Smaller size cells are called microcytic
Larger size cells are called macrocytic
The calculation is done by dividing the hematocrit by the total RBC count, eg:-

HCT % X 10 = fL
RBC in 1012/L

The mathematical derivation of the units of the indices (femtoliter and picogram) are very
complex.
Femptoliter (fL)= 10-15/Liter

MCV (fl) = hematocrit ÷ rbc count

Say your HCT results are 41.3% and your red cell count is 4.32 x 10 12/L:-
MCV (fl) = 41.3% ÷ 4.32 x 1012/L
Then your MCV will be MCV = 95.6fl
Normal range;-- Healthy individual (range) = 80-100 fL
A low MCV means the red blood cells are microcytic.-
a) Iron deficiency anaemia,
b) Lead poisoning.
c) Genetic diseases
Thalassemia major and thalassemia minor - an inability to
manufacture sufficient quantities of globin chains.
Sideroblastic anaemia - an inherited disorder that prevents red
blood cells from making sufficient hemoglobin.
d) Copper deficiency.
e) Vitamin deficiency eg. B6 and C
f) Low stomach acid (Hypochlorhydria) - hampers the uptake of
certain minerals and vitamins.
g) Rheumatoid arthritis.
h) Haemolytic anaemia -.
i) Having one's spleen removed (splenectomy)
A high MCV means the red blood cells are macrocytic.

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a) Hereditary anaemia
b) Megaloblastic anaemias which include
Pernicious anaemia
Folic acid deficiency
Vitamin B12 deficiency - pernicious anaemia
c) Reticulocytosis )
d) Diseases or disorders such as:-
Aplasia - a condition in which RBC precursors in bone marrow are nearly absent.
Myelofibrosis - fibrosis of the bone marrow.
Hyperglycemia - an increase in plasma glucose.
Cold agglutinins - abnormal proteins in the blood which at high level can cause a form of
anaemia.
Manganese deficiency.
e) Liver disease
f) Hypothyroidism
g) Drugs (anti-convulsants)
h) Treatment with the anti-HIV drugs eg. AZT (Retrovir or Zidovidune).
An MCV within the normal range means the cells are normocytic.
The patient may still present with anaemia.
This may be due to :-
a) An acute haemorrhage.

Mean corpuscular hemoglobin (MCH)

MCH expresses the average weight (content) of haemoglobin in individual RBCs .
This calculation tells you the amount of haemoglobin in a single red blood cell.
It is done by dividing the haemoglobin by the total RBC count, eg:-

Hgb in g/dL X 10 = pg
RBC in 1012/L

Normal levels = Healthy individual (range) = 27-33 pg/Cell

MCH (pg) = hemoglobin ÷ rbc count

Say your Hemoglobin results are 13.9g/dL and your red cell
count is 4.32 x 1012/L:-
MCH (pg) = 13.9 ÷ 4.32 x 10 12/L
Then your MCH will be MCH = 32.2 pg/cell (pg/cell =
picograms per cell)

Increased MCH, termed hyperchromasia, is associated with macrocytic anaemia since larger
cells tend to have more hemoglobin.
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Mean Corpuscular Hemoglobin Concentration (MCHC)
MCHC expresses the average concentration of haemoglobin per unit volume of RBCs

This calculates the average amount of haemoglobin per red blood cell
Or to be more scientific, 'the average haemoglobin concentration per unit volume (100 ml) of
packed red cells'.
A low MCHC means that a 100ml of packed red blood cells contain less haemoglobin than
normal and a high MCHC means that there is more haemoglobin in 100ml of packed red blood
cells.
The calculation is done by dividing the haemoglobin by the hematocrit, eg:-

Hgb in g/dL x100 =%

Hct %

Healthy individual (range) = 32-36%

MCHC (g/dL) = hemoglobin ÷ hematocrit

Say your Hemoglobin results are 13.9g/dL and your hematocrit is 0.413:-
13.9 ÷ 0.413 = 33,65
MCHC = 33.7 g/dL
MCHC is decreased in the same conditions as the MCV, for example:-
Decreased MCHC values, termed hypochromia, may be due to:-
a) Iron deficiency anaemia
b) Thalassemia
c) Blood loss
d) Vitamin B6 deficiency
There is a condition where the MCV and MCHC differ.
This occurs in Pernicious anaemia, where the MCV is high but
the MCHC is normal.
In the disease Spherocytosis, a relatively rare disorder, in which
the RBC's are small and round (rather than the normal
biconcave) and therefore hold more hemoglobin, there is an
increase in the MCHC.