Blood and its components

Dr Pronati Gupta
Growing trophozoites P falciparum
Ring trophozoites P falciparum
Gametocyte P. falciparum
 hemoglobin
• Hemoglobin (Hb or Hgb) is a red color pigment present
in red blood cells (RBCs)
• It comprises Fe2+ and Globin protein
• Hb carries the oxygen from the lungs to the tissues and
CO2 from body tissues to the lungs for excretion.
• Gives Red colour to blood.
• chromoprotein consisting of Globin molecule attached
to 4 red colored Heme molecules.
• Hemoglobin synthesis requires the coordinated
production of Heme and Globin.
PRINCIPLE OF SAHLI’s METHOD / ACID HEMATIN METHOD

• When the blood is added to N/10 Hydrochloric acid (HCl),

the hemoglobin present in RBCs is converted to acid
hematin which is a dark brown colored compound.
• The color of the formed acid hematin complex
corresponds to the Hemoglobin concentration in the blood
• The colour is matched with the standard which is a
reference brown glass given in the Sahli’s apparatus by
diluting with N/10 hydrochloric acid or distilled water until
the color of acid hematin complex match with the color of
the standard.
 REAGENTS REQUIRED FOR SAHLI’s METHOD / ACID HEMATIN
METHOD

• N/10 hydrochloric acid (It is prepared by

diluting concentrated hydrochloric acid 0.98
ml in distilled water and volume is made up
100 ml).
• Distilled water
 APPARATUS & EQUIPMENTS

• Sahli’s Apparatus
– Hemoglobin pipette (0.02 ml or 20 µl capacity)
– Sahli’s graduated Hemoglobin tube
– Thin glass rod Stirrer for Hemoglobin Tube
– Sahli’s Comparator box with brown glass standard
• Spirit swab
• Blood Lancet
• Dry cotton swab
• Pasteur pipette
 PROCEDURE

• ⇒ N/10 Hydrochloric acid is taken in

Hemoglobin tube (has two graduations – one
side gm/dl, and other side shows the Hb
%age), up to the mark 20 – the lowest marking
(yellow marking).
• ⇒ Venous or Capillary blood is drawn up to
20µl mark of hemoglobin pipette exactly.
 Procedure
• For capillary blood draw, boldly prick the tip of
the middle or ring finger with the help of
Blood lancet or pricking needle. Wipe out the
first drop of blood and suck the blood from the
second drop in Hb pipette up to the mark of 20
µl. Fill the Hb pipette by capillary action.
• ⇒ Wipe out the surface of the pipette with the
help of tissue paper/ cotton so that excess
blood may not be added to the Hb tube.
 Procedure
• ⇒ Dispense the blood into N/10 hydrochloric
acid taken in the hemoglobin tube, rinse the
pipette with the same solution and mix
properly with the help of stirrer.
• ⇒ Place the tube at room temperature for 10
minutes for complete conversion of
hemoglobin into acid hematin.
 Procedure
• After the reaction completes, place the Hb
tube in the column in Sahli’s Comparator box
and start diluting the dark brown coloured
compound (Acid Hematin) formed in the Hb
tube using the N/10 HCl or distilled water by
adding drop by drop of it into the solution and
mix with the help of stirrer after each addition.
• ⇒
 Procedure
• This process is done until the endpoint comes
matching the color of standard with the color
of the test.
• ⇒ Once the color is matched with the
standard brown glass, lift the stirrer up and
note down the reading in Sahli’s Hb tube by
taking the lower meniscus in consideration.
• Note: Usually in colored solution, the upper
meniscus is considered for taking the reading
but in this case, it is a transparent color
solution and lower meniscus can be recorded
in order to give the exact reading.
 Procedure
• Now add one more drop of distilled water and mix it
properly with the help of stirrer. If color is still
matching with the standard add another drop till it
matches with the standard and note down the
reading and, if it gets lighter after adding the first
extra drop, it shows reading taken before dilution
was correct. Note down that reading as the final
result.
• ⇒ Reading of this method is expressed in
Hemoglobin gm/dl (gram/100 ml) of blood.
 PRECAUTIONS
• Sahli’s apparatus especially the Hemoglobin
pipette and Sahli’s Hemoglobin tube should be
clean and dry before use.
• ⇒ Suck the blood exactly up to the mark of
20 µl (0.02 ml) and air bubbles should not be
present in the pipette with blood.
• ⇒ Mix well the acid and blood and wait for at
least 10 minutes after adding the blood in acid.
 Precautions
• Add distilled water drop by drop and mix well after
each dilution. Avoid over dilution of the content.
• ⇒ The matching of color should be done against the
natural source of light or electrical tube light (white
light) to avoid any visual errors.
• ⇒ Blood sample and N/10 HCl acid should be taken in
an accurate and precise amount in the Hb tube.
• ⇒ The Hb pipette should be wiped off properly in
order to avoid the excess addition of blood in the Acid.
DISADVANTAGES OF SAHLI’s METHOD / ACID HEMATIN METHOD

• ⇒ Visual intensity may be different for different

individuals by this method, we are not able to
measure the inactive hemoglobin.
• ⇒ This method estimates only oxy Hemoglobin.
Carboxyhemoglobin and methemoglobin
cannot be estimated.
• ⇒ The endpoint disappears soon so it is difficult
to know the actual endpoint and also the
Proper stable standard is not available
 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.
• Internationally accepted reference standard calibrator.1
• It is time-consuming and cyanide-dependent protocol
gives it a higher relevance as a reference method for
POC hemoglobin devices and analyzer calibration.
 Principle of Cyanmeth method
• When Blood is mixed with a solution of
potassium cyanide, potassium ferricyanide,
and Drabkin’s solution, the erythrocytes are
lysed by producing evenly disturbed
hemoglobin solution.
• Potassium ferricyanide transforms hemoglobin
into methemoglobin, and methemoglobin
combines with potassium cyanide to produce
hemiglobincyanide (cyanmethemoglobin).
 Absorbance at 540nm
• Absorbance of the solution is measured in a
spectrophotometer at 540 nanometers.
Hemoglobincyanide has a wide absorbance
peak at this wavelength.
• The absorbance is compared with that of the
standard hemiglobincyanide solution by using
a formula to obtain the amount of
hemoglobin.
 Requirements
• Equipment
• A spectrophotometer or photoelectric colorimeter
• Pipette 5 ml
• Sahli’s pipette
• Reagents
• Drabkin’s Solution
• Cyanmethemoglobin standard solution with a
known hemoglobin value
 Procedure
• Take 5 ml of Drabkin’s solution in a test tube and add 20 μl of blood.
• Dilution is 1:25.
• Now mix the mixture and allow to stand for at least 5 minutes.
• This time is adequate for the transformation of hemoglobin to
hemiglobincyanide.
• Pour the test sample into a cuvette and read the absorbance of the
test sample in a spectrophotometer at 540 nanometers or in a
photoelectric colorimeter using a yellow-green filter.
• Also, read the absorbance of the standard solution. Absorbance
must be read against Drabkin’s solution.
• From the formula given below, the hemoglobin value is derived.
• Hemoglobin in gm/dl = [Absorbance of test sample ÷ Absorbance
of standard] x concentration of standard x [Dilution factor ÷ 100]
 Preparation of table and graph
• The result can be obtained quickly if the table of a graph is prepared which
corresponds to absorbance with hemoglobin concentration.
• This is markedly acceptable when a huge number of samples are daily
processed on the same instrument.
• For the preparation of a calibration graph, cyanmethemoglobin standards are
commercially available.
• As another option, a standard cyanmethemoglobin solution is diluted serially
with Drabkin’s solution.
• The concentration of hemoglobin (horizontal axis) in each dilution is arranged
against the absorbance (vertical axis) on a linear graph paper.
• A straight line connecting the points and passing through the origin is
obtained.
• A table can be prepared relating absorbance to the concentration of
hemoglobin from the help of this graph.
 Non invasive Hb detection
• Occlusion spectroscopy is a noninvasive
measurement technology featuring a ring-
shaped sensor that is attached to the subject’s
finger.
•  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.
 Other older methods
• Alkali hematin method
• Copper sulphate method
 Automated Hb methods
• SLS haemoglobin detection method uses cyanide-
free sodium lauryl sulphate (SLS).
• The reagent lyses red blood cells and white blood
cells in the sample.
• The chemical reaction begins by altering the globin
and then oxidising the haeme group.
• SLS’ hydrophilic groups can bind to the haeme group
and form a stable, coloured complex (SLS-HGB),
which is analysed using a photometric method.
 Automated pocedure
• An LED sends out monochromatic light and by moving
through the mixture light is absorbed by the SLS-HGB
complexes. The absorbance is measured by a photo
sensor and is proportional to the haemoglobin
concentration of the sample.
• Absorption photometric methods are usually influenced
by the turbidity of the sample itself. In blood samples,
turbidity can be caused due to lipaemia or leucocytosis.
By using the SLS-HGB method these interferences can be
minimised due to the effect of the reagent.
 NORMAL VALUES OF HEMOGLOBIN

• Adult Male: 14-16 gm/dl

• Adult Female: 13-15 gm/dl
• Newborn: 16-18 gm/dl
 Raised Hemoglobin Content
• Polycythemia Vera
• Associated with Hypoxia
• Cyanotic Congenital Heart disease
• High Altitudes
• Heavy smoking
• Methemoglobinemia
• Elevated erythropoietin levels
– Tumors of Kidney, Liver, CNS, Ovary etc.
– Renal Diseases (Hydronephrosis & Vascular impairment)
• Adrenal hypercorticism
• Therapeutic androgens
• Relative causes of high hemoglobin content
– Dehydration – Water deprivation, Vomiting, Diarrhea
– Plasma loss – Burns, Enteropathy
 Reduced Hemoglobin Content
• Low Hemoglobin value means anemia caused by the following
conditions
• Leukemia
• Tuberculosis
• Iron deficiency anemia
• Parasitic infections severely in hookworm infection
• Sickle cell anemia
• Thalassemia
• Aplastic anemia
• Hemolytic anemia
• Loss of blood
 Cell counting
• Manual : Improved Neubaeurs chamber
• Automated
– Impedance
– Optical
– Hydrodynamic focussing
 Using the Neubauer chamber
• Sample Preparation
• Depending on the type of sample, a preparation of a dilution with
a suitable concentration should be prepared for cell counting.
Typically, the concentration range for a cell count with Neubauer
chamber is between 250,000 cells / ml and 2.5 million cells / ml.
• An appropriate dilution of the mixture with regard to the number
of cells to be counted should be used. If the sample is not diluted
enough, the cells will be too crowded and difficult to count.
• If it is too dilute, the sample size will not be enough to make
strong inferences about the concentration in the original mixture
 Preparing Neubauer Chamber

• Clean the Neubauer chamber and the cover

slip with 70% EtOH. Put the glass cover on the
Neubauer chamber central area. Use a flat
surface to place the chamber, like a table or a
workbench.
 Introducing the sample into the Neubauer
chamber
• With a pipette, carefully draw up around 20 ml
of the cell mixture (dilution). Place the pipette
tip against the edge of the coverglass and
slowly expel the liquid until the counting
chamber is full.
• Capillary action will help to ensure that the
counting chamber is full, but care should be
taken not to overfill the chamber. A volume of
10 ml is sufficient to fill one counting chamber.
Microscope focusing and Cell Counting
• Place the Neubauer chamber on the microscope stage. Using the
10X objective, focus both onto the grid pattern and the cell
particles.
• As 10X is appropriate for WBC counting, count the total number
of cells found in 4 large corner squares.
To count the RBCs and Platelets, the microscope must be
switched to 40X objective. Count the cells in the respective areas
as stated early.
• Write down the amount of cells counted
• If cells are touching the 4 perimeter sides of a corner square, only
count cells on 2 sides, either the 2 outer sides or 2 inner sides.
 Calculating the cell counts
• The total number of cells per microliter of sample can be
calculated from the number of cell counted and area counted.
This is because the ruled areas of the chamber contain an exact
volume of diluted sample.
• Since only a small volume of diluted sample is counted, a general
formula must be used to convert the count into the number of
cells/microliter.
• The dilution factor used in the formula is determined by the
blood dilution used in the cell count. The depth used in the
formula is always 0.1mm.
• The area counted will vary for each type of cell count and is
calculated using the dimensions of the ruled area.
WBC DILUTING FLUID
 Platelet diluting fluid
• 1% Ammonium oxalate
• Lyses RBC and is isotonic to platelets
• WBC remain but number is low and size very
big so does not interfere in counting
• Counted in 1x 1mm central sqaure
Eosinophil diluting fluid
• Centrifuge at 1500rpm for 30 min
HEMATOCRIT
Microhematocrit
Microhematocrit reader
Red cell indices
MCH
MCHC