Hematology

1. CHAPTER 9
HEMOGLOBIN STRUCTURE, SYNTHESIS,
FUNCTION AND MEASUREMENT

2. Acknowledgements
 Addisa Ababa University
 Jimma University
 Hawassa University
 Haramaya University
 University of Gondar
 American Society for Clinical Pathology
 Center for Disease Control and Prevention-Ethiopia

3. Objectives
At the end of this chapter the students will be able to:
 Define hemoglobin
 Diagrammatically illustrate the structure of hemoglobin
 Discribe the biosynthesis of heme and globin moieties of
hemoglobin
 Explain the functions of hemoglobin
 State the principles of hemoglobin estimation in clinical
practice
 Explain the principle of the cyanmethemoglobin method
of hemoglobin determination
 Carry out calibration for the cyanmethemoglobin method
of hemoglobin determination

4. Objective cont’d
 Perform hemoglobin quantitation on a sample of blood
using the cyanmethemoglobin method
 List the advantages of the cyanmethemoglobin method of
hemoglobin determination
 Explain the principle of the Hemocue method of
hemoglobin quantitation
 Descrive the advantages and disadvantages of the
Hemocue method
 Perform hemoglobin quantitation on a sample of blood
using the Sahli-Hellige method
 Explain why the Sahli-Hellige method is considered
unreliable

5. Objective cont’d
 Describe sources of specimen collection errors (pre-
analytic) that can cause inaccurate results
 List the sources of error in various hemoglobin
determination techniques
 Discuss reference ranges and the significance of
hemoglobin values on the basis of age and sex
 Explain the correlation between red blood cell count,
hemoglobin and hematocrit results
 Discuss quality control and checks utilized to establish
test validity and prevent erroneous results

6. 9.1 Structure of Hemoglobin
 Hemoglobin is normally present in red cells
 Two primary structures
 Globin
 Heme which is composed of
 Protoporphyrin
 Iron
 The heme structure consists of a ring of C, H and N atoms
called Protoporphyrin IX with an atom of Ferrous ( Fe2+ )
iron attached ( ferroprotoporphyrin).

7. Basic structure of hemoglobin molecule showing one
of the four heme chains that bind together to form the
Hb molecule

8. Iron
 Iron is an essential component of hemoglobin
 Decreased tissue iron = cellular dysfunction
 Increased tissue iron = cellular destruction
 Regulated by absorption, not excretion
 Iron circulates in the plasma bound to transferrin

9. Iron Compartments/Pools

10. 9.2. Hemoglobin synthesis
 Synthesis of heme and globin moieties proceeds
separately, though not entirely independently
 the process is controlled by feedback mechanism
 e.g., formation of heme increases the synthesis of
globin and lack of heme reduces globin synthesis.
 Heme molecule: a porphyrin ring with an iron atom at
its center (in a ferrous state)
 Porphyrins are tetrapyrroles
 the four pyrroles linked by a methane bridge
 Heme synthesis occurs largely in the mitochondria by a
series of biochemical reactions involving a number of
enzymes and co-factors

11. Protoporphyrin
 Site of synthesis is the mitochondria in
RBC cytoplasm
Protoporphyrin III (9)
HAEME

12. Globin
 Globin chains are composed of amino acids arranged in
a specific pattern
 Site of synthesis is the ribosomes
 4 normal chain types are produced
 Alpha chain composed of 141 amino acid chains
 Beta chain146 amino acid chains
 Gamma
 delta

13. Cont’d

14. Haemoglobin Molecule
Hgb A
α2 β2
Hgb A2
α2 δ2
Hgb F
α2 ϒ2
 Consists of 4 globin chains + 4 heme groups
 heme groups are identical
 Different globin chains determine the hemoglobin type
 3 normal hemoglobin types (by 6 months of age)

15. 9.3 Function of Hemoglobin
 Oxygen binds to central iron atom in heme
 Iron must be Fe2+ (ferrous) state to
transport oxygen
 Each hemoglobin molecule can carry up to
4 oxygen molecules

16. Cont’d
 Two normal Hgb forms
 Deoxyhemoglobin (Fe2+ without oxygen), in
tissues
 Oxyhemoglobin (Fe2+ with oxygen), in lungs
 Two abnormal Hgb forms
 Methemoglobin (Fe3+ ,oxidized)
 Carboxyhemoglobin (Fe2+ with CO)
 Both are reversible

17. HGB/RBC Breakdown
 Aged (1% lost daily) or defective red cells are mainly
removed by splenic macrophages [by reticuloendothelial
system (RES)]

18. 9.4. Methods of Hemoglobin Measurement
(1) Spectrophotometric
a) Cyanmethemoglobin
b) Hemo-Cue
c) Oxyhemoglobin
d) Direct Read- Out
(2.)Visual comparative methods
a)Sahli - Hellige method
b)BMS Hemoglobinometry
( 3) Cu SO4 specific gravity
•Is the measurement of concentration of Hgb in red
cells (whole blood)
•Hgb is reported in g/dL
There are different methods

19. I. Spectrophotometric
1. Cyanmethemoglobin method
 ICSH recommended reference method
 EDTA anticoagulated whole blood or capillary
samples
 All hemoglobin forms are measured
EDTA
whole
blood

20. Cont..
Principle:
 Blood is diluted in a solution of potassium ferricyanide
and potassium cyanide (Drabkin’s solution). The
potassium ferricyanide oxidizes hemoglobins to
hemiglobin (Hi: methemoglobin) and the potassium
cyanide provides CN -- ions to form hemiglobin cyanide
(HiCN) which has a maximum absorption at 540nm.
Finally absorbance of the solution is measured in a
photometer or spectrophotometer at 540nm and
compared with that of a standard HiCN solution

21. Reagent
 Drabkins solution
 Potassium Ferricyanide -
(Hexacyanoferrata)=K3Fe(CN)6
 Potassium cyanide
 Potassium dihydrogen phosphate
 Highly poisonous; store securely in locked cupboard
in light opaque container wrapped in silver foil

22. Procedure
1. Pipet 20l of well mixed anticoagulated blood into 5ml
Drapkin’s solution (1:251)
2. Mix well and allow to stand at room temperature for at
least 5-10 minutes in the dark
3. The absorbance is measured against reagent blank at
540nm.
4. The absorbance of an aliquot of HiCN standard is
measured at the same wavelength
Hb (g/dl) = At  Cst  DF
Ast  1000

23. Calibration
 The Hemoglobin Standard is offered as a dry vial
containing a standardized amount of methemoglobin
prepared from human hemoglobin.
 Reconstituting the Hemoglobin Standard yields the
Cyanmethemoglobin Standard solution.
 The solution will yield an absorbance equivalent to that
of whole blood sample containing a hemoglobin level of
18g/dl that has been diluted 1:251 with Drabkin's
solution.
 Dilutions of the Cyanmethemoglobin Standard Solution
with Drabkin's solution are used to prepare a calibration
curve as follows:

24. calibration curve

25. Reference range:
 Adult males: 13-18g/dl
 Adult females: 11-16g/dl
 Newborns: 14-23g/dl
 Note: reference values vary with age, sex, physiologic
condition, altitude, etc. Thus local reference values
should established.

26. Advantages:
 Stable Hemiglobincyanide standard available to calibrate
instrument
 Convenient method
 Readily available and stable standard solution (readings
need not be made immediately after dilution)
 All forms of hemoglobin except sulfhemoglobin (SHb)
are readily converted to HiCN.

27. Sources of error when measuring
Hemoglobin photometrically
 Not measuring the correct volume of blood due to poor
technique or using a wet or chipped pipet.
 When using anticoaglulated venous blood, not mixing the
sample sufficiently.
 Not ensuring that the optical surfaces of a cuvet are clean
and dry
 Air bubbles in the solution to be measured
 Wrong wavelength
 Improper instrument calibration
 Reagent exposed to light

28. Sources of error cont’d
 Lipemia
 Extremely high WBC count causes cloudiness
 Presence of abnormal Hemoglobins
 Presence of abnormal proteins
Note:
 Lipemia causes an increase in the Hb result due to
cloudiness in the solution read by the spectrophotometer.
 In lipemia, centrifugation can clear the specimen and the
supernatant reading will be accurate.
 Abnormal Hemoglobins or proteins are not lysed by the
reagent, so again the solution is cloudier which makes the
instrument read the Hemoglobin result higher than it is.

29. Haemolyzed plasma
(haemolysis)
Normal plasma
Lipemic plasma
(lipids)
Icteric plasma
(bilirubin)
Hemoglobin Interferences

30. 2. Hemoglobin - HemoCue®
 HemoCue® photometer
 Uses dry reagent system (cuvetes)
 Determines concentration of azide methemoglobin
photometrically
 Electronic check and whole blood control samples
must be run to monitor instrument function and
reagent

31. Principle
 The hemoglobin concentration in a fresh capillary or
anticoagulated blood sample (EDTA preferred) is
determined photometrically using a dry reagent system.
The red cells are lysed and hemoglobin is converted to
azidemethemoglobin by sodium nitrite and sodium
azide. This method of Hb measurement is a widely used
point-of-care test.

32. HemoCue® cont’d
 Procedure
1.Turn on HemoCue® instrument
2. Run electronic calibration check (red control cuvette)
3. Fill specimen cuvette with EDTA or capillary blood in a
continuous process without bubbles.
4. Place cuvette in instrument, insert to ‘measure’ position
 Hemoglobin result will be displayed in g/dL

33. Hemoglobin - HemoCue®
Electronic
calibration red
cuvette
Specimen cuvette

34. Hemocue Cont’d
 Advantage HemoCue® system
 No dilution necessary
 The instrument reads the result when it is ready (no
need to let stand for lysing of RBCs) and result is
reported directly eliminating errors in reading from a
calibration chart
 High accuracy
 No expensive instrument needed
 Disadvantage:
 Test cuvettes are expensive
 Finger prick technique must be good

35. Sources of error HemoCue® method
 Failure to properly collect the blood sample if done as
a capillary collection
 Blood not collected from a free flowing finger prick
 Failure to fill cuvette properly
 If not, read within 10 minutes
of collection

36. Quality control
 Spectrophotometer/photometer
 Whole blood control must be performed
 Performed in duplicate; should match within
plus/minus 0.5 g/dl
 Calibrator for making a standard curve
 HemoCue®
 Calibrator cartridge
 Whole blood control

37. 3.Oxyhemoglobin Method
 The simplest and quickest method for general use with a
photoelectric
 Colorimeter but no longer widely used.
Method:
 A 1:251 dilution of blood is made with 0.007N NH4OH
with thorough shaking to ensure mixing and oxygenation
of Hb.
 The absorbance of the solution is read at 540nm in a
photo-/spectrophotometer against a 0.007N NH4OH
solution as a blank.
 Disadvantage:
 Lack of a stable oxyhemoglobin standard.
 Does not measure carboxyhemoglobin, hemiglobin or
sulphhemoglobin

38. II. Visual comparative method
 Is not recommended because of its unacceptable
imprecise and inaccurate
 Principle
 20 L of blood is mixed in a tube containing 0.1mol/l HCl
which lyses the RBC and converts the hemoglobin to acid
hematin. After 10 minutes ( or more ), 0.1mol/l HCl or
water is added drop by drop, with mixing , until the color
of the solution matches the color of the glass standard
positioned alongside the dilution tube and the
concentration is read from the graduated scale on the
dilution tube
1. Sahli-Hellige

39. Procedure:
1. Fill the graduated tube to the ''20'' mark of the red
graduation or to the 3g/l mark of the yellow graduation
with 0.1N HCl.
2. Draw venous or capillary blood to the 20μl mark of the
Sahli pipet. Do not allow air bubbles to enter into the
Sahli pipet.
 With EDTA anticoagulated venous blood ensure that
it is well mixed by inverting the tube repeatedly for
about 1 minute immediately before pipetting it.
 If using capillary blood, wipe away the first drop of
blood from the finger.

40. Cont..
3. Wipe the outside of the pipet with absorbent paper.
 Check that the blood is still on the “20” mark.
4. Blow the blood from the pipet into the graduated tube
containing the 0.1N hydrochloric acid solution.
5. Rinse the pipet by drawing and blowing out the acid
solution 3 times.

41. Cont..
6. Place the graduated tube in the hemoglobinometer stand
facing a window.
7. Allow 10 minutes for RBC lysis and formation of acid
hematin
8. Compare the color of the tube containing diluted blood with
the color of the standard
 If the color of the diluted sample is darker than that of the
reference, continue to dilute by adding 0.1N HCl or distilled water
drop by drop.
9. Stir with the glass rod after adding each drop.

42. Cont..
10. Remove the rod and compare the color of the tube with
the standard columns.
11. Stop when the colors match.
12. Note the mark reached.
 Depending on the type of hemoglobinometer, this gives
the hemoglobin concentration either in g/dl or as a
percentage of ''normal''.
 To convert percentages to g/dl, multiply the reading by
0.146.

43. Disadvantage
 Fading of the color glass standard and difficulty in
matching it to the acid hematin solution.
 Conversion to acid –hematin is slow
 Because of this, all red cells may not lyse and Hb may
not converted to Acid Hematin in specified lesser time
resulting a falsely decreased Hgb value
 Acid Hematin is unstable
.

44. Disadvantage cont’d
 HbF is not converted to acid hematin and therefore the
Sahli method is not suitable for measuring hemoglobin
levels in infants up to 3 months.
 Interpersonal difference in reading the endpoint of
dilution.
 As a result the Sahli-Hellige method is not recommended
for Hb determination

45. Materials:
 Sahli hemoglobinometer
 Sahli pipet
 Stirring glass rod
 Dropping pipet
 Absorbent cotton
 0.1N HCl

46. 2. Alkaline Hematin Method
 A useful ancillary method under special circumstances
as it gives a true estimate of total Hb even if HbCO, Hi or
SHb is present.
Disadvantage:
 Certain forms of Hb are resistant to alkali denaturation,
in particular, Hb-F and Hb Bart.
 More cumbersome and less accurate than the HiCN or
HbO2 methods and thus is unsuitable for use as a
routine method.

47. 3. BMS Hemoglobinometer
 Requires no dilution of blood
 Use in clinics with a few Hb tests are performed
Principle
 A drop of blood is mixed with a saponin impregnated
stick. This lyses the red cells giving a clear solution of
Hb. The absorption of light by the hemolysed patient
blood sample ( existing in one half of the field of view of
the meter ) is matched with a scale on the meter( that of
the standard in the other half). The Hb value in g/dl is
obtained from a scale on the meter

48. WHO Hb colour scale
 The method is based on comparing the color of a drop of
blood absorbed on a particular type of chromatographic
paper against a printed scale of colors corresponding to
different levels of Hb

49. III. Copper Sulphate Densitometery
 This is a qualitative method based on the capacity of a
standard solution of copper sulphate to cause the
suspension or sinking of a drop of blood.
 The measurement of specific gravity of a sample of
blood corresponds to its hemoglobin concentration.
 The method is routinely utilized in some blood banking
laboratories while screening blood donors for the
presence of anemia.

50. Sources of Error
 Pre-analytical errors are a common cause of inaccurate
results
 wrong patient identification
 improper venous blood sample collection
 Wrong anticoagulant type and concentration
 Failure to mix blood with anticoagulant
 Mislabeling
 improper capillary blood collection
 Clotted sample

51. Cont’d
 Hemoconcentration
 Hemodilution
 Hemolysis does not cause hemoglobin error
 Technical errors (failure to adhere to SOP)
 Post analytic errors
Do controls detect specimen collection errors?

52. Quality Control
 Control samples monitor the correct functioning of
equipment, stability of reagents and testing technique
 Controls do not detect specimen collection errors
 Control samples with known assayed values are used to
check result reliability
 Controls detect invalid results caused by errors in
testing technique, reagents or instrument malfunction

53. X3=15.1
X3=45.0
Tips
 RBC count – total # of red
cells in millions/uL
 Hemoglobin –
concentration of Hb in red
cells reported in g/dL
 Hematocrit – percentage
(%) of red cells in a
known volume of whole
blood
 RBC count, Hb and HCT
values are correlated to
each other
/3=15.1

54. RBC Count, Hb, HCT
 Each health institution should establish its own reference
ranges
 Significance
 Decreased RBC, HGB and/or HCT values….Anemia
 Decreased production, increased loss/destruction
 Increased RBC, HGB and/or HCT
values….Polycythemia
 Increased production
 Critical values: Hb <7.0 or >18.5 g/dL

55. Exercise: Result Evaluation
3 adults
 Patient # 1: All results are normal
 Patient #2: Anemia (and leukocytosis)
 Patient # 3: Polycythemia/erythrocytosis

56. Which parameter does not correlate?
RBC = 4.00 million/cmm
Hb = 14.0 g/dl
HCT = 36.0%
 Hb error; RBC and HCT correlate
RBC Count, Hb, HCT
Correlation
 Relationship: Hb x 3 = HCT + 3%
RBC x 3 = Hb or RBC x 9 = HCT
 Used to estimate values or check data correlation
 ‘Rules’ only apply if red cells are normal in size and hgb
content

57. Summary
 Biochemistry of the hemoglobin molecule
 Cyanmethaemoglobin method (or modifications) of
hemoglobin determination, including specimen
requirements
 Sources of specimen collection error (pre-analytic) that
can cause inaccurate results
 Potential sources of error when measuring hemoglobin
photometrically (i.e., substances that interfere with
photometric measurements)
 Quality control and checks utilized to establish test
validity and prevent erroneous results

58. Review Questions
1. Describe synthesis of the heme and globin moieties of
hemoglobin
2. Summarize the functions of hemoglobin in the body.
3. What are the two most commonly applied color
comparison methods for measurement of hemoglobin in a
sample of blood? Write the test principle of each of these
methods.
4. Compare and contrast (in terms of accuracy, advantage,
drawbacks, etc.) the two routine methods of hemoglobin
quantitation.
5. What is the clinical implication of hemoglobin
measurement in a sample of blood?

59. Review Questions cont’d
6. List at least five pre-analytic errors and their remedies in
Hb determination
7. List at least five possible sources of error and their
remedies in Hb determination using photomertic methods