HEMATOLOGY 1

MLS 413 | LECTURE | MIDTERM

HEMOGLOBINOMETRY PART 1 AND 2

LEARNING OBJECTIVES • Selection of blood donors
• Discuss the different methods of determining o Hemoglobin estimation is a practical test to be
hemoglobin concentration. done among prospect donors prior to blood
• Explain the principle of different ways of hemoglobin donation
determination NOTE: WHO defines anemia as Hb levels of <12 g/dL in
• Identify the advantages and disadvantages of each women and <13 g/dL in men
method of hemoglobin determination VARIATIONS IN REFERENCE VALUES
• Give the reference values of hemoglobin level for HEMOGLOBIN CONCENTRATION VARIATIONS
children and adults. • Hemoglobin concentration varies
• List precautions to be observed when using Drabkin’s • Variation of the hemoglobin concentration is actually
reagent influenced by physiological factors or by
• Understand the importance of preparing the pathological factors
hemoglobin standard curve in the PHYSIOLOGICAL FACTORS
Cyanmethemoglobin method • Age
• Know how to prepare hemoglobin standard • Gender
concentration curve by cyanmethemoglobin method. • Pregnancy
HEMOGLOBIN DETERMINATION/HEMOGLOBIN • Physical activity or Exercise
ESTIMATION o Exercise training can increase total hemoglobin
• Done separately or as part and red cell mass which enhances oxygen
of routine CBC carrying capacity of the person
• Relatively simple o The possible underlying mechanism are
• Performed quickly by the proposed to come mainly from the bone marrow
laboratory including stimulated erythropoiesis with
hyperplasia of the hematopoietic bone marrow
INDICATIONS FOR HB ESTIMATION o Hyperplasia – there is an increase in the number
• The measurement of blood hemoglobin is one of the of cells in a particular organ as in this case the
most common clinical laboratory tests bone marrow
• Hemoglobin estimation is used to indirectly evaluate o In the bone marrow, the hematopoietic cells
the oxygen carrying capacity of the blood; this makes increase in number and that is hyperplasia
it an important aid in detecting and evaluating blood o Also, there in an improvement of the
loss and diagnosing and treating anemia hematopoietic micro environment induced by
• Determine presence and severity of anemia exercise training
• Screening for polycythemia o hormone and cytokine accelerated erythropoiesis
o Screen for blood disorder particularly may come into play that’s why there is an
polycythemia unexpected hemoglobin concentration variation
o Polycythemia – blood disorder associated with during exercise training
an increased red blood cells in the body • Posture (from lying to sitting/standing)
▪ such increase in the red blood cells can o A shift from supine to sitting or standing position
cause the blood of the patient to be viscous would result to hemoglobin concentration
and to be thicker which in turn would result to variation
a complication which is blood clot formation o Posture may influence the concentration of
• Response to specific therapy in anemia several blood constituents due to the decreased
o Evaluate the response of the patient to specific plasma volume occurring on changing from lying
therapy in anemia to standing position
o if not that, then somehow hemoglobin estimation o it is conventionally assumed that remaining
in the laboratory is performed supine for a long time may be associated with
consistent hemodilution
• Estimation of red cell indices
o hemodilution – increase in the fluid content of
o One of the parameters used to calculate or use
the blood resulting in diminution of the
to compute for the red blood cell indices or
concentration of formed elements
erythrocyte indices

FIRST SEMESTER 1
HEMATOLOGY 1
MLS 413 | LECTURE | MIDTERM

HEMOGLOBINOMETRY PART 1 AND 2

o The standing posture may be a cause of blood o The increase in hemoglobin level of blood in
concentration due to the effect of gravitational smokers could be a form of compensatory
force and hydrostatic pressure. This will cause mechanisms.
ultrafiltration of the plasma and the small PATHOLOGICAL FACTORS
molecules in the interstitial space • Testosterone Deficiency
• Altitude o Also known as hypogonadism
o The percentage of oxygen in the air at 3.2 o Prevalent in men with chronic kidney disease
kilometers is essentially the same at sea level. wherein the testosterone, supposedly, stimulates
However, the air pressure is 30% lower at the erythropoiesis via the production of
higher altitude. E.g Mt. Apo, Mt. Everest. This is hematopoietic growth factors and possible
due to the fact that the air there is less dense improvement of iron bioavailability.
o Air molecules at higher altitude are farther apart o It is expected that it would affect the stimulation
making it less dense. So, when we breathe in air of erythropoiesis.
at sea level, the atmospheric pressure of about • Renal Deficiency
14.7 lbs/sq. in (pounds per square inch) • Myelodysplasia
causes the oxygen to easily pass through the o Also known as myelodysplastic syndrome
selective permeable lung membranes into the o It develops because the bone marrow cells do not
blood smear develop into mature blood cells. So, what
o However, at higher altitude, the lower air pressure happens?
makes it more difficult for oxygen to enter the ▪ Instead, these blood cells stay within the
vascular system resulting now to hypoxia or bone marrow in an immature state. If
oxygen deprivation immature, it cannot serve its function,
o Hence, the body needs to respond to this resulting in low blood cell count.
particular stimulus (hypoxic stimulus) and that is • Diminished Erythropoiesis
to increase erythropoiesis and alongside with it is o Less stimulation of RBC production, resulting in
a rapid increase also in the concentration of decrease hemoglobin concentration.
hemoglobin. So this provides mountaineers with • Bm Suppression
a means to compensate for the dramatic fall in the o It cannot facilitate erythropoiesis properly.
arterial oxygen saturation seen at high altitude • Blood Loss
o The final concentration of hemoglobin vary o Specially if it’s a massive blood loss.
o The increase is largely dependent upon the NORMAL HEMOGLOBIN CONCETRATION IN
altitude reached by the person and the HUMANS
individual’s arterial oxygen saturation • Men – 150 ± 20g/L or 13g/dL
• Smoking • Women (not pregnant) – 135 ± 15 g/L or 11-16 g/dL
o Increase in hemoglobin concentration is believed • Pregnant women
to be mediated by exposure of carbon monoxide o Another physiological factor affecting hemoglobin
and in fact some scientist suggested that concentration is pregnancy
increase in hemoglobin level in blood of smokers o Recall that red blood cells are primarily formed in
could be a form of compensatory mechanism the bone marrow and their production is highly
o The carbon monoxide binds to the hemoglobin to affected by pregnancy
form carboxyhemoglobin, an inactive form of o Hemoglobin concentration varies at different
hemoglobin. stages or different gestational ages during
▪ This does not have an oxygen carrying pregnancy
capacity o 1st trimester: 124-135 g/L
o So, the carboxyhemoglobin also shifts the o 2nd trimester: 110-117 g/L
hemoglobin dissociation curve in the left side o 3rd trimester: 106-109 g/L
resulting in the reduction in the ability of o NOTE: Pregnancy significantly decrease the
hemoglobin to deliver oxygen to tissues hemoglobin concentration of woman wherein the
o To compensate the decrease oxygen delivering hemoglobin concentration along with the
capacity, smokers maintain a higher hemoglobin hematocrit and red blood cell fall during
level than non-smokers.
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HEMATOLOGY 1
MLS 413 | LECTURE | MIDTERM

HEMOGLOBINOMETRY PART 1 AND 2

pregnancy because the expansion of the C. Gasometric Method
plasma volume is greater than that of the red CYANMETHEMOGLOBIN METHOD
blood cell mass • Most accurate method for estimation of Hb
o However, there is a rise in a total circulating • Recommended by International Committee for
hemoglobin directly related to the increase in red Standardisation in Hematology (ICSH) and National
cell mass Committee for Clinical Laboratory Standards (CSLI)
o this in turn depends partly on the iron status of a because:
pregnant woman that’s why pregnant women are o All forms of Hb are converted to
recommended to have a hemoglobin level cyanmethemoglobin (except sulfhemoglobin)
between 12-16 g/dL o Stable and reliable standard is available.
o any value below 12 is considered as iron CYANMETHEMOGLOBIN METHOD – EQUIPMENT
deficiency but not yet anemia • Photoelectric colorimeter (Right) or
o Hb value below 10.5 is considered as anemia spectrophotometer (Left)
• Birth – 180 ± 40 g/L (high hemoglobin concentration) • Sahlis pipette at 20 micro litre
o In other text reference book (Rodak’s): 15-20 o Design for sipping blood sample for hemoglobin
g/dL regardless of the gender of the baby determination
o ↓ to 10-14 g/dL by 2mos of age • Pipette 5 ml
o 12-15 g/dL by 10 years of age
• In females, hemoglobin level reaches a plateau
during early puberty while in males, the hemoglobin
level continues to rise throughout puberty to higher
levels characteristic of adult men
• Men and women have different mean hemoglobin
levels; it is probably a direct effect of sex hormones REAGENTS REQUIRED
both estrogen and androgens on erythropoiesis
DRABKIN’S SOLUTION
• However, since there is no difference in erythropoietin
• pH of the solution: 7:0-7.4
levels between the sexes this effect most likely takes
o pH must be maintained in order not
place in the kidney rather than in bone marrow
to alter the result of hemoglobin
HEMOGLOBIN MEASUREMENT IN THE
• Unstable if exposed to light
LABORATORY
(Photosensitive)
• Blood Can Be Collected From 3 Different Sources:
o Deteriorates once exposed to light
1. Capillary Blood
• Stored at room temperature in brown borosilicate
2. Venous Blood
bottles
▪ Preferred sample: EDTA Anticoagulated
o Stored in amber bottle or brown borosilicate bottle
blood, Heparinized tube (Rodak’s)
• Solution should be clear and pale in yellow color
3. Arterial Blood
o Examine macroscopically the quality Drabkin’s
solution.
o The transparency should be clear, and the color
is pale yellow.
o If there’s a change in the Drabkin’s solution, it
must not be used. Otherwise, it will alter the
hemoglobin concentration result
• Composed of various solutions, such as:
METHODS OF HB ESTIMATION
o Potassium ferricyanide
A. Visual Methods
o Potassium cyanide
o Specific Gravity
o Sodium bicarbonate
o Sahli’s Acid Hematin
o Surfactant
o Tallquist Hemoglobin Color Chart
HB STANDARD SOLUTION
o WHO Hemoglobin Color Scale
• Part of the Cyanmethemoglobin Method reagent kit
B. Photoelectric Methods
along with Drabkin’s solutiion
o Cyanmethemoglobin method
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HEMATOLOGY 1
MLS 413 | LECTURE | MIDTERM

HEMOGLOBINOMETRY PART 1 AND 2

• Available commercially at specific • Mix blood with Drabkin’s and wait for 5 to 10 minutes
concentration for the reaction to take place.
• Stored in a brown bottle as it is o Ensure that the pipette tip is devoid with blood.
photosensitive o Alongside, prepare a standard solution with a
• Exposure to light causes deterioration in determined hemoglobin concentration.
the strength of the standard
o Much more so this than Drabkin’s solution
• The concentration of the standard used in this
demonstration is 14.8 gm/dL
• Other commercially available standard solution for
hemoglobin is available at 15.1 gm/dL concentration
CYANMETHEMOGLOBIN METHOD – SPECIMEN
[On right] Representation of the methodology. Prepare
• EDTA anticoagulated blood two test tubes with 5 mL Drabkin’s solution (One for the
• Capillary blood blank without blood – left; One with blood –
CYANMETHEMOGLOBIN METHOD right).
• Measure the absorbance of the sample
against blank (Drabkin’s reagent) at 540
nm by spectrophotometer.
CYANMETHEMOGLOBIN PROCEDURE
(ACTUAL PHOTOS)
• Step 1 – Transfer the reagent. Two test tubes are
being prepared but this preparation still varies. For
example, five
patients are being
• Principle examined for
o (1) Blood is diluted in alkaline Drabkin solution. cyanmethemoglobin
Erythrocytes are lyse producing an evenly procedure. So,
distributed hemoglobin solution number of test tubes
o (2) The Potassium Ferricyanide converts the should be increased.
hemoglobin molecule → Methemoglobin • Step 2 – Add 20 uL of
(oxidized form of hemoglobin; also known as specimen to the test tube with
hemiglobin) Drabkin’s solution labelled as
o (3) Methemoglobin + Potassium Cyanide → ‘Test.’ Make sure there is no
Cyanmethemoglobin which forms a stable- blood clinging to the tip of the
colored compound pipette. If the 20 uL of blood
o (4) Absorbance is measured in a colorimeter or is not completely dispensed
spectrophotometer at 540nm in the Drabkin’s solution, then it would alter the
• Stable compound cyanmethemoglobin hemoglobin concentration.
o Also called hemiglobincyanide • Step 3 – Stand for 10 minutes to allow full conversion.
▪ Because this is a result of the hemiglobin Some textbooks say 5-10 minutes. Some say fixed 10
complex with Potassium Cyanide. minutes. The blood added with a reagent. Potassium
METHODOLOGY ferricyanide will convert the hemoglobin (which is
• Add 5 mL of Drabkin reagent to a test tube. released by the red blood cells) into methemoglobin
o Prepare two test tubes. Each test tube contains 5 (oxidized form of hemoglobin; also called as
mL of Drabkin’s solution. Hemiglobin). Another component of the Drabkin’s
reagent which is Potassium cyanide will be oxidized
• On the test tube with Drabkin’s solution labeled ‘Test,’
Add 0.2 mL or 20 uL blood to the test tube using Hb the Methemoglobin. Then, it would be converted into
Cyanmethemoglobin (more stable; hemoglobin with
pipette or micropipette
o The other test tube with Drabkin’s solution will cyanide; hemiglobincyanide) upon the action of
stand as ‘Blank.’ Potassium cyanide.

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HEMATOLOGY 1
MLS 413 | LECTURE | MIDTERM

HEMOGLOBINOMETRY PART 1 AND 2

o Check the concentration of the standard stamped
on the ampule or vial.
o In this case, it is 59 mg/dL.
59 mg/dL → gm/dL
𝟓𝟗
= 𝟏𝟎𝟎𝟎
= 0.059 gm/dL
o The value now needs to be adjusted to the
• Step 5 – Spectrophotometer. Measure the dilution factor.
absorbance at a 540 nm in spectrophotometer or 20 uL (whole blood) + 5000 uL → 5020 uL
colorimeter. Dilution factor
• Step 5a – Read the 𝟓𝟎𝟐𝟎
=
“Blank” tube (540 𝟐𝟎

nm; Absorbance: = 251

0.00; Transmittance: o Using cyanmethemoglobin, the dilution is 1:251.
100%). o The value needs to be adjusted to the dilution
• Step 5b – Read factor.
specimen tube o Multiply 0.059 x 251 = 14.809
(refers to the sample ▪ Concentration of the standard in gm/dL =
with Drabkin’s 0.059
solution and blood). ▪ Dilution factor = 251
Record absorbance o We are taking the concentration of standard to be
reading. used in the calculations as 14.8.
• Step 6 – Compute for Hemoglobin.
Concentration of Unknown Specimen:
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝑼𝒏𝒌𝒏𝒐𝒘𝒏
X Concentration of Standard
𝑨𝒃𝒔𝒐𝒓𝒃𝒂𝒏𝒄𝒆 𝒐𝒇 𝑺𝒕𝒂𝒏𝒅𝒂𝒓𝒅
(g/dL)
o The concentration of standard is incorporated in
the package insert, oftentimes, it is 14.8 g/dL or
15.1 g/dL.
PREPARATION OF STANDARD CURVE FOR
HEMOGLOBIN ESTIMATION BY
CYANMYETHEMOGLOBIN METHOD
• The standard curve is prepared by using
• The first thing we need to do is make serial dilutions
cyanmethemoglobin standard solutions of known
of the standard solution with that of the Drabkin’s
hemoglobin concentrations. This preparation
solution in several test tubes.
standard curve is an important exercise to
• Notice in test tube 1, it is the only test tube that
standardize the test method. This demonstration uses
contains the Drabkin’s solution (5mL) without a
14.8 g/dL.
Standard solution. So, the dilution factor would be 0
Hb ESTIMATION BY CYANMETHEMOGLOBIN
or the dilution is 0/5.
METHOD
• The 2nd test tube is filled with 1mL of the Standard
• REAGENTS REQUIRED
solution and 4mL of the Drabkin’s solution making a
o Available commercially at a specific
1/5 dilution or a dilution factor of 0.2.
concentration
• The 3rd test tube is filled with 2mL of the Standard
o Stored in a brown bottle as it is photosensitive
solution and 3mL of the Drabkin’s solution making a
o Exposure to light causes deterioration in the
2/5 dilution or a dilution factor of 0.4.
strength of the standard.
• The 4th test tube is filled with 3mL of the Standard
o The concentration of the standard used in this
solution and 2mL of the Drabkin’s solution making a
demonstration is 14.8 gm% or 14.8 g/dL
3/5 dilution or a dilution factor of 0.6.
• Deriving the Standard Value

FIRST SEMESTER 5
HEMATOLOGY 1
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HEMOGLOBINOMETRY PART 1 AND 2

• The 5th test tube is filled with 4mL of the Standard • For some procedures, you can measure it directly
solution and 1mL of the Drabkin’s solution making a using a test tube (picture on the right, colorimeter).
4/5 dilution or a dilution factor of 0.8. But for most laboratories, they use the cuvettes.
• Test tube 6 contains only the Standard solution (5mL) • Cuvettes can be made from either glass or quartz.
and not the Drabkin’s solution. So, the dilution factor This is used to measure a solution in a
would be 1 or the dilution is 5/5. spectrophotometer.
• In a colorimeter, place the test tube as seen on the
picture above and set the density to 0 and the
absorbance at 540nm wavelength. Measure the
optical density of each dilution in the
spectrophotometer or in colorimeter always against a
blank reagent or the blank solution (purely Drabkin’s
solution).
• Every time we measure, we must use a clean cuvette.
OPTICAL DENSITY OR ABSORBANCE

• The picture above shows the preparation of the serial

dilution.

• After measuring the absorbance of each sample, we

record the optical density or the absorbance.
ABSORBANCE VALUE

• Strength of the standard = 14.8 gm/dL

• Value of Hb = dilution factor X Strength of the
standard
o In test tube 1: 14.8 X 0 = 0 (0%)
o In test tube 2: 14.8 X 0.2 = 2.9 (3%)
o In test tube 3: 14.8 X 0.4 = 5.9 (6%)
o In test tube 4: 14.8 X 0.6 = 8.9 (9%)
o In test tube 5: 14.8 X 0.8 = 11.8 (12%)
o In test tube 6: 14.8 X 1.0 = 14.8 (15%)
CALIBRATION

• This time we are now ready to plot a graph. The

optical density or the absorbance value should be in
the y-axis or the vertical axis while the concentration
of the hemoglobin should be in the x-axis or the
horizontal axis.
• This could be plotted in excel or in a graphing paper.
• A straight line passes through the origin in an upward
• After deriving the value of hemoglobin, it is now time
manner. So, that line is what we call the standard
to measure the absorbance.
curve of hemoglobin.
FIRST SEMESTER 6
HEMATOLOGY 1
MLS 413 | LECTURE | MIDTERM

HEMOGLOBINOMETRY PART 1 AND 2

HEMOGLOBIN STANDARD CURVE • Use of dirty or scratched cuvettes
o Always handle the cuvette with care and check
the cuvettes that we are using prior to use since
it can also alter the results.
• Use of deteriorated reagent
o Deteriorated reagents lead to erroneous results.
DRABKIN’S REAGENT
• Appearance – clear and pale yellow
• pH – 7/0-7.4
• Bottle – sensitive to light
• The picture above shows another example of a o Uses amber bottle or brown borosilicate glass or
hemoglobin standard curve. It is plotted in a graphing stored in a dark place
paper. • Temperature – room temperature
o >30 degrees Celsius (refrigerate)
o Cool down first before using if stored in a
refrigerator
• Cyanide – highly toxic. Must be used cautiously
o Discard it in a sink with running water
CONDITIONS THAT AFFECT Hb DETERMINATION =
FALSE POSITIVE
• Specimen
o Turbidity
• This is a clearer picture of hemoglobin standard
▪ Centrifuge first for 5-10 mins
curve. The graph on the right is based on the values
▪ May be due to high WBC and platelets
presented on the left.
o Hemolysis
A NEW CALIBRATION CURVE MUST BE PREPARED
▪ Hemolyzed specimen must be diluted first to
WHENEVER
1:2 with distilled water and multiply the result
to 2
o Lipemic
▪ Centrifuge first, then obtain 0.01 mL of
plasma, then add 5mL of Drabskin’s reagent
and read as blank
• Reagent precipitation
o Due to abnormal proteins
o Plasma cell myeloma and macroglobulinemia
• Hemoglobin Type
o In smokers, carboxyhemoglobin is obtained from
their blood. The rate of conversion of the blood is
• If another reagent lot is used for a specific test, then slow
you really need to prepare a new standard calibration o The reaction is prolonged to 30 mins to 1hr to
curve for hemoglobin. overcome the problem
• This is to ensure the reliability and accuracy of the ADVANTAGES
hemoglobin concentration that the laboratory is • All forms of Hb except sulphhemoglobin are
reporting. converted to
MECHANICAL SOURCES OF ERROR hemoglobincyanide/cyanmethemoglobin (HiCN)
• Pipetting error • Visual error us not there was no color matching is
o Too much or too little amount of volume required
transferred is a source of error since it alters the • Cyanhemoglobin solution is stable and its color does
results. It could either lead to false increase or not fade with time so readings may not be taken
false decrease of hemoglobin. immediately

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HEMATOLOGY 1
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HEMOGLOBINOMETRY PART 1 AND 2

• Absorbance may be measured soon after dilution. • Ex. (pic on the right) A
• Absorbance and stable reference standard are blood sample amounting
available from World Health Organization for direct to 2cc or it could vary
comparison (amounts of blood may
• Stable reference standard considered by ICSH and vary from 0.1 to 2cc).
CLSI. Within those blood
DISADVANTAGES volume that can be used
• Diluted blood has to stand for a period of time to for this method
ensure complete conversion of Hb • Hemoglobin from the blood is determined by
• Potassium cyanide us a poisonous substance and measurement of the capacity of hemoglobin to
that is why Drabkin’s solution must never be pipetted combine with carbon monoxide
by mouth • The blood must be saturated with carbon monoxide
• The rate of conversion of blood containing and the subsequent analysis is caried out in the
carboxyhemoglobin is slowed considerably. monometric gas apparatus of Van Slyke and Neil
Prolonging the reaction to 30mins can overcome this (they are the ones who devised this apparatus)
problem. • Oxygen carrying capacity measured by Van Slyke
• Abnormal plasma proteins cause turbidity when blood apparatus
is diluted with Drabkin’s solution • Based on formula, 1 gm of Hb carries 1.34 ml of
• A high leukocyte count also causes turbidity on oxygen
dilution of blood. Centrifuging the diluted blood can • It does not measure:
help overcome turbidity o carboxyhemoglobin
PART 2 - METHODS OF HB ESTIMATION o Sulfhemoglobin
• Since most hemoglobin reagents contain hazardous o Methemoglobin
chemicals such as cyanide or azide like the ones • Time consuming and expensive
used in cyanmethemoglobin method, care must be o Although this is a good method to estimate the
taken when performing the test hemoglobin concentration. However, it is time
• Different precautions to be observed include wearing consuming and expensive
gloves, working in a well-ventilated area, proper • Result is 2 percent less than other methods
disposal of used reagents (e.g cyanide; has to be o The hemoglobin result when analyzed in this
flushed or discarded with running water), wiping up of apparatus is 2% less than other methods
spills on the working area, and handwashing SPECIFIC GRAVITY METHOD/GRAVIMETRIC
I. VISUAL METHODS METHOD
• Specific Gravity • The method or technique of measuring hemoglobin is
• Sahli’s Acid Hematin not routinely performed except as a method of
• Tallquist Hemoglobin Color Chart screening blood donors
• WHO Hemoglobin Color Scale • Rough estimate is made from specific gravity of blood
II. PHOTOELECTRIC METHOD • Copper sulfate technique
• Cyanmethemoglobin method o Makes use of copper sulfate solution
o The most recommended method for • Used in mass screening like selection of donors
hemoglobin determination by ICSH and CLSI • Rapid and simple
III. GASOMETRIC METHOD o Rapid and simple but, it gives only an estimate of
GASOMETRIC METHOD hemoglobin concentration and it requires no
• This method is based on the capacity of hemoglobin special instrument so you’re going to use only
to combine with the same maximum volume of carbon copper sulfate solution with no special instrument
dioxide as of oxygen to be used
• The whole process involves saturation of the blood o How are we going to perform such? We are going
with carbon monoxide to perform capillary puncture on the skin of the
person and then a drop of blood either directly
from the skin or collect it first in a capillary tube
and then drop it in the copper sulfate

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HEMOGLOBINOMETRY PART 1 AND 2

o A drop of blood is allowed to fall into a copper ▪ Pipette
sulfate of a particular density or specific gravity ▪ Colour comparator
• Commonly used in blood donor selection MATERIALS FOR ACID HEMATIN METHOD
• A drop of blood is allowed to fall in copper sulphate
solution of specific gravity of 1.053 from a height of
1 cm
• Specific gravity is equivalent to 12.5 grams/dL
• Drop of blood gets covered with copper proteinate
and remains separate and distinct for about 15-20
seconds
• If drop sinks within 15-20 seconds, specific gravity is
higher than copper sulfate
o Copper sulfate is lower than the specific gravity of
blood sample • The picture above shows the different materials in a
o Approximate value of hemoglobin is about 12.5 Sahli apparatus
g/dL • [Pink Box] Sahli’s pipette or Sahli’s hellige
o And the hemoglobin level is then acceptable for with a graduation marks of 20 μL
blood donation o Actual Sahli’s pipette (Right picture)
• If a drop of blood sample floats on the surface, the o (1) Sip blood after doing capillary
hemoglobin value is less than 12.5 g/dL and so the puncture
donor is temporarily deferred; unacceptable for blood o (2) Remove excess blood
donation o (3) Sip the succeeding drops of blood in
a Sahli’s hellige pipette up to 20 μL
• [Red Box] Graduated hemoglobin tube
o Graduations are in two forms (Left – %;
Right – gm%)
• [Yellow Box] Glass rod or stirrer
• [Orange Box] Comparator
o It is where the graduate
hemoglobin is placed and
compared the color of the solution with the
comparator on the sides.
COMMON SOURCES OF ERROR IN SPECIFIC
o Brown glass standards
GRAVITY METHOD
• Sahli Tube for blood
1. Hypergammaglobulinemia (e.g., multiple
• Microtube of Sahli pipette
myeloma)
• Graduated hemoglobin tube
o Excess of gamma globulins in the patient’s blood
STEPS
o Falsely high Hemoglobin level
• Preparation of the acid solution and later on will
2. High WBC count
produce the hematin molecule
o Falsely high Hemoglobin level
1. Put 0.1 HCl till the mark 10%
3. Air Bubbles
2. Add 0.02 ml blood (using micro tube (Sahli hellige) of
o Falsely low Hemoglobin level
the pipette)
4. Use of inadequate or inappropriate height for
3. Shake for 15 minutes
dropping
o Underestimating the Hemoglobin concentration o Using a glass rod or stirrer
ACID HEMATIN METHOD o The solution will start to change in color → brown
color
• Sahli apparatus
4. HCl + Hb → acid hematin (brown in color)
o Haemoglobinometer or
5. Add distilled water to the acid hematin drop by drop
haemometer) which is
o Why drop by drop?
composed of:
▪ Sahli tube
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▪ To recognize the color change and compare o It should be free from blood.
it properly with the comparator • Withdraw the blood horizontally from the finger
▪ Once drop fully, the color of the solution o Ensure alcohol is completely dried
cannot be compared to the color comparator o Avoid compression of the finger.
6. Match colors at full arm length and against light ADVANTAGES OF ACID HEMATIN METHOD
STEPS • Easy to perform
• Quick
• Inexpensive
• Can be used as a bedside procedure
(1) Putting of the 0.1 HCl
• Does not require technical expertise
till the mark 10%
DISADVANTAGES OF ACID HEMATIN METHOD
● For maximum color, longer time is required
● Perfect matching with brown glass standard is not
(2) Collect capillary possible
blood and sip it using ● Carboxyhemoglobin, methemoglobin and
the Sahli hellige pipette sulfhemoglobin are not converted to acid hematin
up to 0.02 mL ● Development of color is slow and acid hematin is not
(3) Shake and add stable
distilled water drop by o You add distilled water until such time you will
drop until such time the achieve the color that is similar to that of the color
color matches with the comparator.
color comparator ● Source of light will influence the comparison of colors
● Average human body (70%): 1 mol of Mg2+ (24g)
PRINCIPLE
TALLQUIST HEMOGLOBIN CHART
• Adding HCl to the blood causes:
o Hemolysis of RBCs
▪ 0.1 normal HCl acid is a hypotonic solution.
When the red blood cells are exposed to a
hypotonic solution, it will result to hemolysis.
▪ By hemolyzing the RBCs, it will liberate the
hemoglobin molecule from the RBCs. The
liberated hemoglobin molecules are made to
react with HCl acid. Then, it produces acid
hematin.
o Formation of acid hematin • Series of lithographed colors said to correspond to Hb
HCl + Hb → acid hematin (brown color) values ranging from 10 to 100 percent
• By adding distilled water per drop, at 12 • Blood obtained from finger puncture
gm%, the color is the same with the color
• Placed on a piece of absorbent paper
comparator.
• Color is matched against the color on the chart
▪ The intensity of color is proportional to
• Corresponding reading taken
hemoglobin content in blood
• Cheap and simple
THE ROLE OF HCL IN DETERMINATION OF HB
CONTENT • Error-20 to 50 percent
• Somewhat similar to a urine dipstick
• As it is hypotonic results in the hemolysis of RBCs.
• The Tallquist method is a valid method for screening
• Convert the hemoglobin to a brownish acid hematin.
anemia especially among pregnant women. This is
PRECAUTIONS
particularly used among women in resource-poor
• Be sure that there are no air bubbles in the blood
settings even in rural primary healthcare centers. An
column.
example would be in Southwest Nigeria.
o Or else, it will result in erroneous result.
• Clean the tip of pipette from any blood to avoid false
high results.
FIRST SEMESTER 10
HEMATOLOGY 1
MLS 413 | LECTURE | MIDTERM

HEMOGLOBINOMETRY PART 1 AND 2

WHO HEMOGLOBIN COLOUR SCALE o SDS
• Devised by Scott and Lewis • Measurements are made at various wavelengths
• Principle is similar to Tallquist method depending on final stable product
• Rapid, simple, inexpensive, reliable • Absorbance measurements are made at a set time
o Same with Tallquist method interval after mixing the blood and the active
• 1g/dL for diagnosis of anemia reagents, but before the reaction is complete.
• Printed set of colors corresponding to Hb values from • The cyanide-free reagent is ideal for use on an
4 to 14g/dL automated high through-put clinical hematology
• Efficiency is greater than 90% detecting anemia analyzer.
• 86% in classifying its grade
• Useful for screening blood donors
• Screening women and children in health programs
• Monitors iron-therapy
• Procedure:
o (1) Obtain blood
sample from the skin
via capillary puncture

o (2) Blot a drop of

blood on the special
absorbent paper

o (3) Insert the blotted

paper into the
different set of colors.
Match the color

• Result:
o Color of the acquired
blood corresponds to
the Hb. Thus, it
indicates the Hb
value.

AUTOMATED METHOD
• Modification of cyanhemoglobin method
• Other chemicals:
o SLS
o Imidazole

FIRST SEMESTER 11