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

 

¾ Neutrophil alkaline phosphatase score is low, but

normal in aplastic cases

¾ Hypercoagulability

¾ Direct Coombs’ test ±.

Further Investigations

¾ Hemoglobinuria

¾ Hemosiderinuria

¾ Serological tests.

• 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

4 Concise Book of Medical Laboratory Technology: Methods and Interpretations Laboratory Diagnosis of Lead Poisoning

Blood Picture

¾ Basophilic stippling in peripheral smear and bone

marrow

¾ Normocytic, normochromic or microcytic, hypochromic

anemia

¾ Polychromasia, slight reticulocytosis and erythroblastemia

¾ Osmotic fragility of red cells is decreased

¾ TLC is usually normal.

Bone Marrow

¾ Stippling of erythroblasts

¾ Sideroblastosis.

Urine

¾ Increased excretion of coproporphyrin and ALA.

Laboratory Diagnosis of Hemoglobin

Structure and Synthesis Disorders

Clinical finding

Ethnic origin of patient Suggest hemoglobinopathy

Any family history

Preliminary blood Hemoglobin electrophoresis

studies confirms

Special Tests

Tests Depending on Physiochemical Properties

¾ Sickle test, Hb solubility tests—HbS

¾ Intracellular Hb crystals—HbC

¾ HbH inclusions—Thalassemia

¾ Heinz bodies

¾ Heat instability test

¾ Isopropanol precipitation test—Unstable Hb

¾ Oxygen dissociation studies—High O2 affinity Hb

¾ Alkali denaturation, acid elution test—HbF.

Hemoglobin Electrophoresis

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

studies.

FIG. 9.19: Electrophoretic band separation

Clinical Hematology 255

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

(Fig. 9.20).

Features

¾ Quantification of five classical bands of serum protein

by default

¾ N-Band (2–15) to quantify any pattern

¾ Accepts any multiband pattern on a transparent dry

media

¾ Postscan EDIT facilitie

¾ Patient identification with date and time

¾ Hard copy of densitogram and/or computed values

¾ Optional ‘PC-LINK 205’ software.

Minimum System Configuration

¾ Systronics Densitometer 205

¾ 80 column dot-matrix printer (EPSON compatible).

System Configuration with ‘PC-:LINK 205’ Software

¾ Systronics Densitometer 205

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

color monitor.

Technical Specifications

Optical

Wavelength range: 400–700 nm

Filters: 520 nm, 600 nm

(interference) and white

light; another

up to 5 filters optional

Light source: 6 V, 6 W tungsten lamp

Slit size (projected): 0.5 mm × 7 mm

Detector: Silicon photodiode

Density range: 0 to 2 OD

Mechanical

Scanning length: 120 mm, programmable

 in steps of 0.1 mm

Scanning speed: 40 mm per minute

Carriage movement: X-axis: automatic

 Y-axis: manual.

Scanning resolution: 10 readings per mm

Maximum pattern size: L-120 mm, W-50 mm,

 H-5 mm

Electronics

Microprocessor: 8085 (Intel)

Display: 1. Segment LED display

for set-up/programing

interface.

 2. 80-column dot-matrix

printer for display of:

 a. Densitogram (with

graduated X and Y axes

to facilitate editing)

 b. Computed values

 c. Patient ID No.

 d. Date and time.

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

program keys: soft-touch

Computations: Proportional fraction in

 % and values with

 reference to total for:

 1. Five classic bands serum

proteins (by default).

 2. N-band (2 to 15, selectable).

Editing: 1. Baseline shifting

 2. Deletion of area.

 3. Selection of minimas

(rejection or introduction).

Printed Records

1. 5-band Serum: a. Proportional fraction %

Protein mode of albumin, alpha-1,

 alpha-2, beta, gamma

 b. Protein values of albumin,

alpha-1, alpha-23, beta,

gamma with reference to

total protein value

 c. Albumin/globulin ratio

OR

N-band mode: a. Proportional fraction % of

N-bands

 b. Values with reference to

total

2. Patient ID No: Up to 999.

3. Date and time: D/M/Y/and Hr/min.

Power

230 V ± 10%, 50 Hz.

Optional Accessories

i. RS 232 interface to communicate with external

computer

ii. ‘PC-LINK 205’, software.

Laboratory Diagnosis of Sickle Cells Trait

1. Sickling tests (positive).

2. Solubility tests for HbS (positive).

3. Hb electrophoresis.

Laboratory Diagnosis of Sickle Cell Anemia

Blood Picture

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

erythropoiesis

• Hemolytic and aplastic crises further reduce Hb

concentration

• 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

Blood Picture

1. Mild to severe anemia:

• Hemolytic anemias

• Peripheral blood

• Anisopoikilocytosis

• Punctate basophilia, polychromasia

• Reticulocytosis.

2. MCH is reduced

3. Red cell life is 20–30 days.

Special Tests

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

body formation.

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

pH = 8.6) in 50% cases.

THALASSEMIAS (REDUCED SYNTHESIS RATE)

Thalassemias are of two types:

¾ a, affecting mainly a chain

¾ b, affecting mainly b chain.

In b Thalassemia

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

in cells forming aggregates → causing ineffective

erythropoiesis.