¾ Neutrophil alkaline phosphatase score is low, but
• Ham’s serum test: Patient’s red cells will undergo lysis
in compatible acidified serum at 37°C (serum may be
patient’s own). About 10–50% lysis implies a positive
test with patient’s own serum.
• Sucrose hemolysis test: Isotonic solution of low ionic
strength if causes hemolysis of more
¾ Basophilic stippling in peripheral smear and bone
¾ Normocytic, normochromic or microcytic, hypochromic
¾ Polychromasia, slight reticulocytosis and erythroblastemia
¾ Osmotic fragility of red cells is decreased
¾ Increased excretion of coproporphyrin and ALA.
Laboratory Diagnosis of Hemoglobin
Structure and Synthesis Disorders
Ethnic origin of patient Suggest hemoglobinopathy
Preliminary blood Hemoglobin electrophoresis
Tests Depending on Physiochemical Properties
¾ Sickle test, Hb solubility tests—HbS
¾ Intracellular Hb crystals—HbC
¾ Isopropanol precipitation test—Unstable Hb
¾ Oxygen dissociation studies—High O2 affinity Hb
¾ Alkali denaturation, acid elution test—HbF.
The apparatus consists of two basic units:
1. The electrophoresis trough.
2. The voltage and amperage regulator. There are two
troughs on either side containing the appropriate fluid.
The paper strips or agar gel covered slides are placed in
the center of the instrument, and either end is connected
to the respective trough with the help of wet filter paper
strips—with one end dipping in the fluid and the other
touching the gel or paper strip. This kind of connection is
made on both the sides. The hemolysate is applied gently
with the help of coverslip (broken) to get about 0.5–1
cm size. The positive and negative points are connected
to the electrophoresis trough and then it is covered.
Electrophoresis is allowed to continue for a specific time.
The agar gel slides are dipped in amido black and left
overnight for destaining in dilute acetic acid solution. The
hemoglobin bands become clearly visible, the agar slide
can be left for drying and a permanent record obtained.
Having used the apparatus once, change the anode and
cathode terminals for use next time. Most electrophoresis
troughs are provided with water cooling systems, attach
the inlet tube to a slow water stream from a tap and the
outlet water to be drained into a waste sink.
Cellulose acetate electrophoresis done at pH = 8.6. Agar
gel electrophoresis, using citrate buffer done at pH = 6.0. The
mobilities of electrophoresis are presented in Figure 9.19.
(For further precise quantitations, electrophoresis can
be done on starch gel or starch block)
Autoscanning and Computing Densitometer 205
Densitometer 205 is microprocessor based, designed to
quantify electrophoretically separated bands (Fig. 9.19).
The embedded software allows quantification of serum
protein pattern by default, and any other multiband
pattern (e.g. hemoglobin, lipoprotein, etc.) in an N-BAND
mode. Postscan facilities like base line shifting, deletion of
area, selection of minimas make the unit practically very
useful and enhance its application in research-oriented
FIG. 9.19: Electrophoretic band separation
A special ‘PC LINK 205’ software (optional) links
the Densitometer 205 to a PC. With this, densitograms
can be conveniently stored/recalled/displayed/edited/
compared on a CRT monitor using the keyboard of the PC
¾ Quantification of five classical bands of serum protein
¾ N-Band (2–15) to quantify any pattern
¾ Accepts any multiband pattern on a transparent dry
¾ Patient identification with date and time
¾ Hard copy of densitogram and/or computed values
¾ Optional ‘PC-LINK 205’ software.
¾ 80 column dot-matrix printer (EPSON compatible).
System Configuration with ‘PC-:LINK 205’ Software
¾ 80 column dot-matrix printer (EPSON compatible)
¾ Systronics PC-LINK 205 software
¾ PC (286, 386, 486 or Pentium or higher) with VGA/EGA
Light source: 6 V, 6 W tungsten lamp
Slit size (projected): 0.5 mm × 7 mm
Scanning length: 120 mm, programmable
Scanning speed: 40 mm per minute
Carriage movement: X-axis: automatic
Scanning resolution: 10 readings per mm
Maximum pattern size: L-120 mm, W-50 mm,
Display: 1. Segment LED display
FIG. 9.20: Representative image of electrophoresis and densitometer apparatus
256 Concise Book of Medical Laboratory Technology: Methods and Interpretations Computing and Editing
Input of date and 11 numerical and 13 functional
Computations: Proportional fraction in
2. N-band (2 to 15, selectable).
1. 5-band Serum: a. Proportional fraction %
Protein mode of albumin, alpha-1,
N-band mode: a. Proportional fraction % of
3. Date and time: D/M/Y/and Hr/min.
i. RS 232 interface to communicate with external
Laboratory Diagnosis of Sickle Cells Trait
2. Solubility tests for HbS (positive).
Laboratory Diagnosis of Sickle Cell Anemia
1. Anemia—moderate to marked; normocytic, normochromic (MCV and MCH are normal).
• Anemia occurs because of reduced RBC lifespan
to about 8 days and in part due to ineffective
• Hemolytic and aplastic crises further reduce Hb
• Irreversibly sickled cells (ISC) have a life of 2 days
• Osmotic fragility is decreased
2. ESR is decreased, sickling prevents rouleaux formation.
3. There is moderate to marked anisopoikilocytosis.
4. Serum and red cell folate values are decreased.
5. There is evidence of intravascular hemolysis.
6. Sickle and solubility tests are positive.
7. There is irregular distribution of HbF (acid elution test).
8. Hemoglobin electrophoresis shows increased HbS.
Laboratory Diagnosis of Unstable Hemoglobinopathy
• Punctate basophilia, polychromasia
3. Red cell life is 20–30 days.
1. Demonstration of Heinz bodies: Preformed Heinz
bodies cannot be shown by supravital staining except
in 50% of splenectomized patients. Sterile incubation
of affected red cells at 37°C for 24 hours leads to Heinz
2. Heat instability test: Incubate a fresh hemolysate
with phosphate or tris buffer at 50°C for 1 hour—
precipitation implies presence of unstable Hb.
3. Isopropanolol precipitation test: Incubate fresh
hemolysate with isopropranolol tris buffer at 37°C for
1 hour, precipitation denotes presence of unstable Hb.
4. Hemoglobin electrophoresis: Abnormal Hb band (at
THALASSEMIAS (REDUCED SYNTHESIS RATE)
Thalassemias are of two types:
¾ b, affecting mainly b chain.
There is reduced b chain production hence reduced HbA
leading to microcytic, hypochromic anemia. Total Hb is
maintained by increase in γ and d chains so HbA2 and HbF
increase. Because of lack of b chains, a chains accumulate
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