MBBS Practical-PATHOLOGY

INSTRUMENTS

Dr Sulekha Singh
1. Urinometer
2. Sahli’s hemoglobinometer
3. Westergren Pipette
4. Wintrobe tube
5. Neubauer Chamber
6. RBC Pipette
7. WBC Pipette
8. Bone Marrow needles : Salah needle, Jamshidi needle
9. Lumbar Puncture Needle
10. Anti sera for Blood grouping
11. Vacutainer tubes/ Vacuum tubes
12. Microtome knife
13. Tissue cassette
14. Paraffin block
15. Leuckart ‘L’ mould
 1. URINOMETER

- Urinometer is a specialized hydrometer an instrument used to measure the

specific gravity of urine.

- It consists of a weighted glass cylinder which floats in urine and has a calibrated
stem to measure specific gravity at a given temperature. The scale in urinometer
read in small divisions from 1.000 to 1.060

- It is usually calibrated in the range of 15° to 20°C (depends on the manufacturer).

As the specific gravity varies with temperature, the reading obtained should be
corrected to the room temperature by applying temperature correction.
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Temperature correction: For every 3°C rise in room temperature beyond the
calibrated temperature (15°C), add 0.001 to the recorded reading; and subtract
0.001 for every 3°C fall in temperature.

Correction for protein and sugar: Subtract 0.003 for every 1 g/dL of protein and
0.004 for every 1 g/dL of glucose

Method:
1. Fill 2/3rd of cylinder with urine.
2. Lower urinometer gently into the urine and let it float freely.
3. Let urinometer settle; it should not touch the sides or bottom of the cylinder.
4. Take the reading of SG on the scale (lowest point of meniscus) at the surface
of the urine.
5. Take out the urinometer and immediately note the temperature of urine with a
thermometer.
6. Adjust the reading by applying temperature correction.

Technique when only small volumes of urine are available:

The urine may be diluted with equal or double its volume of distilled water.
The specific gravity is calculated as follows:–
Dilution with equal volume = Last two figures of the observed value × 2
Dilution with double volume = Last two figures of the observed value × 3.
Normal Specific Gravity of Urine - 1.003-1.030

Other methods to measure SG

1. Reftractometer method
2. Reagent strip method

Causes of Increase/ decrease in SG of Urine

 2. SAHLI’S HEMOGLOBINOMETER

It is used to estimate hemoglobin in blood.

Methods of hemoglobin estimation

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Baddow
Apparatus
It consists of:
• Hemoglobin pipette which has one mark of 0.02 mL (20 cu mm).
• A comparator which has a flat surface and a standard brown color.
• A square/round tube with markings of hemoglobin percentage (100% = 14.5 gm/
dL) on one side and hemoglobin (in gm) on the other side.
• Glass rod for mixing the blood in the square/round tube.
• A dropper to take and transfer N/10 HCl and distilled water into the square tube.

Sahli’s Method or Acid Hematin Method

- This is the most popular colorimetric method.

- Principle: When blood is added to N /10 HCl, hemoglobin is converted into acid
hematin. The resulting brown color of acid hematin is matched against the
standard brown color of the comparator. The intensity of the brown color
depends on the amount of acid hematin produced, which in turn depends on the
amount of hemoglobin in the blood sample.
- Technique
• Take N/10 HCl with a dropper into the hemoglobinometer tube up to the lowest
mark.
• Draw blood up to 20 cu mm (0.02 mL) mark of Hb pipette and wipe off the
excess blood at nozzle of the pipette with moist cotton. Blood may be taken
either from a finger prick or an EDTA vial.
• Blood in the Hb pipette is blown into the square tube of hemoglobinometer.
Ensure that the entire blood inside the Hb pipette is transferred to the tube.
• Wait for 10 minutes for the acid to act on the RBCs which causes lysis of RBCs
liberating hemoglobin. The acid converts Hb to acid hematin. (About 95% of Hb is
converted at the end of 10 minutes, 98% of the color develops at the end of 20
minutes and the maximum color is reached after about 1 hour).
• With the help of a glass rod, mix the acid hematin.
• Match the color of the solution in the tube with that of the comparator by
holding against the natural light or fluorescent tube light.
• If the solution in the tube is darker, add distilled water with a dropper and stir
the solution with a glass rod. This process is continued till the color of the
solution exactly matches the color of the comparator.
• Take the tube out and remove the glass rod. Take the reading from the lower
meniscus.
• Hemoglobin is recorded as gram/dL (or gm%).

- Sources of error
A. Technical errors: These are errors inherent in handling the sample.
- Improper collection
- Improper mixing
- Insufficient time for formation of hematin
- Time delay in taking the reading results in errors.

B. Visual errors:
Comparison of colors is very subjective and can vary from person
to person. Hence, the results may not be accurate.

C. Errors inherent in the equipment

- The accuracy of different equipments is not uniform. If Hb pipette has not
been accurately calibrated at 0.02 mL it can give rise to wrong values.
Calibration of pipettes will reduce the errors.
- Color comparators can affect the reading. If the glass blocks of the
comparators are old or faded, it may give an inaccurate value.
Disadvantages of the method
- Apart from the above technical errors, it is not an accurate method.
- Hydrochloric acid does not convert all hemoglobins (like carboxyhemoglobin,
methemoglobin and sulphemoglobin) into acid hematin. Thus, Hb value obtained may
be slightly lower than the actual Hb.
- Non-hemoglobin substances such as protein, lipid and cell stroma interfere with
the color of blood diluted with acid and hence give false results.

Advantages of the method

- Simple and easy method
- Cheap.

Normal range Depends on the instrument used

Males : 13-17 gm/ dL
Females : 12-15 gm/dL
Newborns : 14-19 gm/dL
 3. WESTERGREN PIPETTE

- It is used to estimate ESR.

- Westergren pipette is a straight glass pipette (open at both ends) 30 cm in length,
bore of 2.55 mm and calibrated in millimeters from 0-200. The capacity of tube
is about 1 ml.
- Method:
SPECIMEN: Venous blood is collected in trisodium citrate solution in 4:1 (blood:
citrate) proportion.
Alternate anticoagulant is EDTA (1.5 mg/ml); however, such anticoagulated blood
sample should be diluted with trisodium citrate just before the test (1 volume of
trisodium citrate to 4 volumes of blood).

PROCEDURE:
1. Mix anticoagulated blood sample thoroughly. The Westergren pipette is filled with
the blood sample up to the “0” mark. A rubber bulb or a mechanical device
should be used for filling. There should be no air bubbles in the blood.
2. The tube is placed in a strictly vertical position in the ESR stand and left
undisturbed for 1 hour. It should not be kept in direct sunlight and should not be
subjected to vibrations.
3. After exactly 1 hour, read the height of the column of plasma above the red cell
column in mm.
4. Express the result as:
Erythrocyte sedimentation rate = ——— mm in 1 hour.

Advantage: This is a more sensitive and accurate method when compared to the
Wintrobe method. This is because the column is longer, i.e. 200 mm.

Precautions 1. Use the correct proportion of blood and anticoagulant. Mix blood and
anticoagulant thoroughly. There should be no clots and air bubbles in blood.
2. The reference range relates to test performed at room temperature of 18-25°C.
If temperature is higher, ESR will increase and different reference range will have
to be derived.
3. ESR tube must be kept vertically. Even a slight tilting will increase the ESR.

Erythrocyte Sedimentation Rate by Westergren Method:

- Males < 50 years: 0-15 mm in 1 hr
- Females<50 years: 0-20 mm in 1 hr
- Children: 0-10 mm in 1 hr
- Elderly (>50 years): Males: 0-20 mm in 1 hr and females: 0-30 mm in 1 hr.
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 4. WINTROBE TUBE

- Wintrobe tube can be used first for estimation of ESR and then it is centrifuged
for estimation of hematocrit.

- Length of Wintrobe tube is shorter (110 mm) & internal diameter is about 3 mm.

- Advantages
• It is simple and requires small amount of blood.
• Same tube can be used for both ESR and PCV.
• Buffy coat smear is useful in cases of leukopenia, subleukemic/aleukemic
leukemia and for LE cell test.

- Wintrobe’s method is more reliable when ESR is low, while Westergren’s method is
more sensitive for high ESR.

- Erythrocyte Sedimentation Rate by Wintrobe Method

• Males: 0-7 mm in 1 hour
• Females: 0-15 mm in 1 hour
• Children: 0-6 mm in 1 hour
 ESR

- The erythrocyte sedimentation rate (ESR) measures the rate of settling

(sedimentation) of erythrocytes in anticoagulated whole blood.

- STAGES OF ERYTHROCYTE SEDIMENTATION RATE : There are three stages of

erythrocyte sedimentation:
Stage 1: Formation of rouleaux or lag phase (10 minutes): Red cells stack together
like a pack of coins. The ESR depends mainly on this stage.
Stage 2: Sinking of rouleaux or decantation phase (40 minutes): Rapid and constant
sedimentation. The longer the tube, the longer is this stage and higher the value of
ESR.
Stage 3: Packing of rouleaux (10 minutes): Slow sedimentation.

- FACTORS AFFECTING ERYTHROCYTE SEDIMENTATION RATE

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Causes of Decreased ESR

• Polycythemia vera
• Sickle cell disease
• Hypofibrinogenemia

Alternative Methods
• Capillary method (micro ESR method) requires only 0.2 mL of blood, so it is useful in
pediatric patients. The results are precise and reading time is 20 minutes.
• Zeta Sedimentation Rate
• Automated ESR (e.g. Ves-Matic benchtop analyser): Results are obtained within 30
minutes and are comparable with Westergren 1 hour reading.
 5. HEMOCYTOMETER/ NEUBAUER CHAMBER

- Hemocytometer: The hemocytometer is an instrument used for counting blood

cells. The commonly used instrument is the improved Neubauer counting chamber.

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- There are two ruled areas on the surface of the chamber. Each ruled area is 3
mm × 3 mm in size and consists of 9 large squares with each large square
measuring 1 mm × 1 mm.
- When the special thick coverglass is placed over the ruled area, the volume
occupied by the diluted blood in each large square is 0.1 ml.
- In the improved Neubauer chamber, the central large square is divided into 25
squares, each of which is further subdivided into 16 small squares. A group of 16
small squares is separated by closely ruled triple lines

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 6. RBC PIPETTE

RBC pipette (Thoma red cell pipette):

- It consists of a graduated capillary tube divided into 10 parts marked 0.5 at the
fifth mark and 1.0 at the tenth mark
- The capillary tube opens into a mixing bulb containing a glass bead which facilitates
mixing of the blood and the diluent.
- There is another short capillary tube above the bulb marked 101.
- Pipette is provided with an aspirating tube.
- In the red cell pipette, blood is drawn up to the 0.5 mark and diluent up to the
101 mark. The resulting dilution is 1:200.

Uses of the RBC pipette

1. RBC count
2. Platelet count.
3. WBC count when total count is very high.

RBC diluting fluid:

1. Hayem’s fluid
2. Dacie’s fluid

Uses of the (red/white) glass bead in

the bulb of (RBC/WBC) pipette:
1. Identification of pipette (red in
RBC and white in WBC pipette).
2. Helps in mixing blood sample
with diluting fluid.
3. Ensures that the pipette is dry.
If pipette is wet,the corresponding
bead will be sticking to the wall
of the pipette.
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Causes of Increased RBC Count

Causes of Decreased RBC Count

• Anemias
• Hemodilution
 7. WBC PIPETTE

- The size of the pipette is smaller than that of RBC pipette and it has a white glass
bead in the bulb which facilitates mixing of the blood and the diluent.
- It has 3 markings of 0.5, 1 and 11.
- In the white cell pipette, blood is drawn up to the 0.5 mark and diluent up to the
11.0 mark.
- The dilution in this case is 1:20.

Uses of the WBC pipette

1. Manual WBC count
2. Absolute eosinophil count (AEC)
3. Sperm count
4. Any fluid (e.g. Pleural fluid) cell count.

WBC diluting fluid:

Turk’s fluid: constituents - Glacial acetic acid,
1% Gentian violet and distill water.

Normal Range for WBC Count

 Increased WBC Count:
Total leukocyte counts more than 11,000/cu mm (11 × 109/L) is termed as
leukocytosis.

Decreased WBC Count:

Total leukocyte count less than 4,000/cu mm (4 × 109/L) is termed as leukopenia.
 8. BONE MARROW NEEDLES

Salah bone marrow aspiration needle with stilette

Parts of the Salah Bone Marrow Needle

• Stilette: It prevents entry of soft tissue and bone fragments into the aspiration
needle when it pierces through them. The stilette has to be removed when the needle
reaches the marrow space.
• Aspiration needle: For aspirating the marrow, a syringe is attached after removing
the stilette.
• Guard: It adjusts the depth of penetration of the aspiration needle.
Sites for Bone Marrow Aspirate
• Anterior superior iliac spine
• Posterior superior iliac spine
• Iliac crest
• Sternum
• Spinous process of lumbar vertebra.

In infants, upper end of the tibia is the ideal site for marrow aspirate.

Indications for Bone Marrow Aspiration

A. Diagnostic
[Link] hematolymphoid disorders
- Red cell disorders: Nutritional anemia (e.g. megaloblastic anemia), pure red cell
aplasia
- White cell disorders: Subleukemic/ aleukemic leukemia, diagnosis and classification
of acute leukemias
- Megakaryocytic disorders: ITP and other thrombocytopenias
- Myeloproliferative neoplasms: Polycythemia vera, chronic myeloid leukemia,
idiopathic thrombocythemia
- Myelodysplastic syndromes
- Plasma cell neoplasms: Multiple myeloma, Waldenström macroglobulinemia

2. Systemic diseases
- Storage disorders: Gaucher, Niemann-Pick disease
- Staging of lymphoid malignancies and solid tumors
a. Lymphoma
b. Metastatic deposits (e.g. carcinoma prostate, breast, lung, kidney)

3. Detection of infection and/or source of PUO

- Parasitic disorders: Kala-azar
- Fungal disorders: Histoplasma
- Mycobacterial infection

4. Miscellaneous disorders: Pancytopenia or unexplained cytopenias

B. Post-treatment follow up: To know the response to therapy and follow up in cases
of leukemia, aplastic anemia and agranulocytosis.

C. Therapeutic: Bone marrow transplant

Contraindications
1. Hemophilia
2. Congenital hemorrhagic disorders.
 9. LUMBAR PUNCTURE NEEDLE

The needle measures 10-12 cm in length. In children, a shorter needle is used. It

has a needle and a stilette.

Sites:
Lumbar puncture: In adults, CSF is normally collected in the midline of the lower
back in the 3rd lumbar space and in children in the 4th lumbar space.

Indications for Lumbar Puncture

A. Diagnostic Indications
1. Infection
* Meningeal infection:
a. Bacterial
Pyogenic
Tuberculosis
Syphilitic: To differentiate general paresis of insane, tabes
dorsalis and meningeal syphilis.
b. Viral
c. Fungal
* Encephalitis

2. Subarachnoid hemorrhage
3. Primary or metastatic malignancy (e.g. acute leukemia, lymphoma)
4. Demyelinating diseases: Multiple sclerosis and subacute sclerosing panencephalitis
(SSPE), Guillain-Barré syndrome
5. Spinal canal blockage leading to elevated intracranial tension (spinal cord tumors)
B. Therapeutic Indications
1. Spinal anesthesia
2. Intrathecal injection of chemotherapeutic drugs for CNS prophylaxis/relapse of
ALL, lymphomas.

Contraindications for Lumbar Puncture

1. Raised intracranial pressure
2. Local infective lesion
3. Disseminated sclerosis
4. Brain tumor

Complications of Lumbar Puncture

1. Post puncture headache
2. Herniation of cerebellum through the foramen magnum due to raised intracranial
pressure.
3. Hematoma, either extradural or subdural.
4. Introduction of infection by the LP needle through the infected skin or
subcutaneous tissue.
 10. ANTISERA FOR BLOOD GROUPING

Colour-coded antisera bottles for ABO-Rh blood grouping

Blue = Anti A
Yellow = Anti B
Colourless = Anti-D (Rh) grouping.
11. VACUTAINER TUBE / VACUUM TUBES

BARAAT

Toasted
12. Microtome Knife (Plain wedge
Type)

13. Tissue cassette (Metal- Stainless steel)

14. Paraffin Tissue Block

15. Leuckart ‘L’ mould