haemoglobin is very important in blood banking and most performing test in a lab. mostly check anemia.

1. Haemoglobin
By Dr.Shahida Baloch

2. Haemoglobin
The hemoglobin is one of the
protein present in the human blood
(more specifically in the RBCs).

3. Haemoglobin structure
Alpha-
type
Alpha-
type
Beta-
type
Beta-
type
Haem

4. Erythrocytes, Hb, and Oxygen Transport
Nearly all of the O2 carried by whole blood in animals is bound and
transported by hemoglobin (Hb) in erythrocytes (red blood cells).
Erythrocytes lack a nucleus and organelles such as mitochondria and have a
half-life in the bloodstream of 120 days.
.

5. Functions of hemoglobin
Imparts red color to the blood.
Helps to carry out the oxygen and other gases
assisting the respiratory system.
It buffers the blood pH and maintains it to the
tolerable limits.

6. Method of estimation of haemoglobin
 The role of technology in hemoglobin testing has led the way for the creation
of innovative devices and techniques for the quantification of hemoglobin
concentration in patients.
 Using an automated hemoglobin analyzer, for instance, has enabled fast,
accurate, and reliable results in the field of hematology.
 A hemoglobin test may rely on different techniques, including reagent based
and “reagent-less” methods, or a variety of non-invasive methods

7. Measuring Techniques
 Photometric
 Visual comparative technique
 The absorbance of hemoglobin in a blood sample is measured
electronically by a colorimeter or different analyzers.

8. Hemoglobincyanide (HiCN) Method
 Using the principle of hemoglobin conversion
to cyanmethemoglobin by adding ferricyanide
and potassium cyanide, the HiCN method of
hemoglobin measurement can proceed.
 Advantages of this type of hemoglobin test is
the availability of an internationally accepted
reference standard calibrator.
 The HiCN method is still routinely used in
clinics especially in resource poor countries;
however, its time-consuming and cyanide-
dependent protocol gives it a higher relevance
as a reference method for POC(Point of care
analyzer) hemoglobin devices and analyzer
calibration.

9. Vanzetti's Azide Methemoglobin
 Hemoglobin conversion via potassium ferricyanide to the colored, stable
azide methemoglobin form that has an almost identical absorbance
spectrum as HiCN, which represents the key characteristic of Vanzetti’s
azide methemoglobin method.2
 A similar reagent used in the HiCN reference method is used, except a
sodium azide is substituted for the potassium cyanide. This hemoglobin
measurement method shows high specificity and sensitivity.3
 Point-of-care (POC) hemoglobin measurement devices, such as the
HemoCue® 201 and EKF Hemo Control, operate on a modification of
Vanzetti’s method.
 First, blood is pulled into a dry reagent cuvette by capillary action.
Secondly, the reagent destroys the red blood cell walls. Free hemoglobin
is then oxidized to a methemoglobin and finally converted to azide
methemoglobin. The susceptibility of the reagent to humidity represents
a challenge associated with this method.

10. Reagent-Less” Methods
 ReaDue to the limitations of reagent-based cuvettes,
POC devices have been developed to feature “reagent-
less” cuvettes.
 HemoCue® 301 was the first POC device to possess
such a feature. This device quantifies absorbance of
oxygenated and deoxygenated hemoglobin, while
turbidity is measured and compensated for at 880 nm.
 Currently an EKF Diagnostics company, DiaSpect
represents a leader in new technology that measures
hemoglobin without a reagent based on broad spectrum
photometry. The DiaSpect technology flashes a white
LED light through a sample to an optical sensor
component.
 This sensor element identifies absorbance of the blood
at a broad wavelength range that will provide insight
into the overall absorbance spectrum resulting in a
higher specificity and a lower sensitivity to
interferences. “Reagent-less” Diaspect cuvettes
feature extensive durability with total self-life of 2.5
years. Just about one second of measurement time is
another benefit of this type of device.


11. Non-Invasive Methods
 With the availability of new technologies to
detect the spectral pattern and concentration
of hemoglobin, non-invasive methods have
become more frequently used, first
introduced for monitoring during surgery and
lately to provide hemoglobin spot checks in
primary care and in blood donor qualification.
 Some non-invasive devices use pulse oximetry
while others rely on white light and the
capturing of transmission data to measure
hemoglobin concentrations in tissue
capillaries.
 Occlusion spectroscopy is a noninvasive
measurement technology featuring a ring-
shaped sensor that is attached to the
subject’s finger.5 The sensor temporarily
ceases blood flow, initiating an optical signal
which yields a high signal-to-noise ratio. This
provides a measurement of hemoglobin
concentration.

12. Sahli´s Method
 Sahli’s hemoglobinometer is a manual device that
contains a hemoglobin tube, pipette, and stirrer,
as well as a comparator. Hydrochloric acid converts
hemoglobin to acid hematin, which is then diluted
until the color of the solution matches that of the
comparator block.
 The clinician can then ascertain the hemoglobin
concentration by reading from the calibration
tube. Although this is one of the most common
methods for estimating hemoglobin in developing
countries and is relatively simple and inexpensive,
the results are not always precise. For example,
there is often inter-observer variability, and it’s
also highly prone to errors due to manual
pipetting.3

13. Sahli' s Method for Estimation Of Haemoglobin:
Principle :
- This method depends on converting Hb. Into acid haematin ( has a dark brown color ).

14. - Sahli apparatus ( Haemoglobinometer ) consists of :
A- Central graduated tube that have two graduations; one indicating
the amount of Hb. In gm ∕ 100cc ,while the other indicating the percentage.
B- Two standard tubes on its sides.
- Special pipette : of 0.02 ml capacity.
- N∕10 Hcl solution& Distilled water.
- Dropper & Glass rods .
Reagents and equipments :

15. Procedure :
1- Take N∕10 Hcl up to the mark 20 % in the graduated tube with the help of dropper.
with the help of dropper.
2- Withdraw a blood by the pipette up to the mark of 0.02 ml. without air entry in

16. 5- Allow tube to stand for 10 minutes so that Hb is converted to acid
haematin.
6- Add distilled water drop by dop and mix well with a glass rod.
7- continue this process till the color of the contents of the central tube
is the same as in the standard tubes.
8- Record your results by noting the level of diluted blood
graduated tube which indicates the percentage of Hb.
And grams of Hb.∕100cc. blood.

17. Hematology Analyzer
 An automated hematology or hemoglobin
analyzer is commonly used for providing high
throughputs to analyze a variety of red and white
blood cells as well as hematocrit and hemoglobin
levels from the blood sample.
 These analyzers offer higher precision value at a
fraction of the time when compared with manual
methods.
 The initial cost of an automated analyzer is high
and regular maintenance and the laboratory
personnel that are needed for the device can
increase costs. Additionally, stable climate
conditions are required which makes it an
unsuitable option for non-laboratory
environments like mobile blood donor test sites
and anemia screening projects out in the field.

18. Blood Gas Analyzer (BGA)
 Typically used with arterial blood,
BGAs measure the combination of
blood gas, pH, electrolytes, and
metabolite parameters.
 Some laboratories may use BGAs
for hemoglobin testing, but they
are more commonly seen in critical
care units, delivery wards, and
emergency rooms.
 The recent development of ready-
to-use sensor and solution
cassettes with automated
calibrations has made BGAs more
user-friendly and robust, but
maintenance is still required. With
the latest innovation of hand-held
devices operating single use
cartridges BGA has become
available for use in mobile
settings, too.

19. Normal adult Hb
 HbA (alpha2/beta2)
 97% +
 HbA2 (alpha2/delta2)
 2-3%
 HbF (alpha2/gamma2)
 0.5% or less
 NOTE ALL NEED ALPHA!

20. Haemoglobin abnormalities
 Haemoglobinopathies
 normal amounts of abnormal beta chains
 crystalline disorders (S, C, D, E)
 familial polycythaemia, M Hb, unstable Hb, HPFH
 Thalassaemias
 reduced amounts of normal alpha or beta chains
 Can be BOTH!

21. Thalassaemias (simplistic)
 Reduced production of BETA chains
BETA thalassaemias
 Reduced production of ALPHA chains
ALPHA thalassaemias more severe clinical disease

22. Why get tested
Hemoglobin is measured:
To detect Anemia or Polycythaemia and to assess its severity.
To monitor the patients response to treatment.
Prior to donating blood.
To calculate red cells indices.

23. Specimen Required
 Venous Blood
 EDTA anticoagulted venous blood (most common)
 Capillary Blood
 Finger-prick (children and adults)
 Heal-prick (infants)


24. Ways of measuring include:
 HiCN technique
 Direct read out method (DHT)
 Oxyhemoglobin technique
 Automated Analyzer
 Photometric Technique
 Based on matching the color of a patient’s sample against a standard.

25. Ways of measuring include:
 Hemoglobinometer
 WHO Hemoglobin color chart
 Sehli’s acid hematin method
 Result Interpretation

26. Reference ranges:
Children at birth - 13.5-19.5 g/dl
Children (2-5 years) - 11.0-14.0 g/dl
Children (6-12 years) - 11.5-15.5 g/dl
Adult men - 13.0-18.0 g/dl
Adult women - 12.0-15.0 g/dl
Pregnant women - 11.0-13.8 g/dl

27. Result Interpretation
 Anaemia:
 Associated with low hemoglobin level, due to:
 Acute blood loss,
 Decreased erythropoitic activity, or Hemolysis
 Classification: Microcytic Hypochromic
 Normocytic Normochromic
 Macrocytic Normochromic
 Polycythaemia:
 Associated with raised hemoglobin level, PCV and red cell count.
 True Polycythaemia (Inc. in number of red cells) Primary , Secondary

28. Quality Control
 Use of appropriate anticoagulant.
 Sample should be checked of having clot.
 Use of standards and controls.
 Storage and stability.
 Blood samples and controls must be allowed to sit at room temperature prior to
testing.
 Sample rejection criteria:
 Hemolyzed or clotted samples are unacceptable for testing.
 Blood collected in sodium citrate, or in tubes containing a gel separator.
 When using anticoagulated blood, not mixing the sample appropriately.
