INSTRUMENTS

PRACTICAL PATHOLOGY

[Link] BISHT
[Link]
CONTENTS
• SAHLI’S HEMOGLOBINOMETER
• WBC PIPETTE
• RBC PIPETTE
• IMPROVED NEUBAUER’S CHAMBER
• VACUTAINERS
• BONE MARROW ASPIRATION AND BIOPSY NEEDLES
• LUMBAR PUNCTURE NEEDLE
• WESTERGREN’S PIPETTE
• WINTROBE’S TUBE
• URINOMETER
• ESBACH’S ALBUMINOMETER
• PAP SMEAR KIT
• SYRINGE WITH NEEDLE
• FNAC HANDLE
SAHLI’S
HEMOGLOBINOMETER
• Use- to estimate hemoglobin of a given sample
• Other methods of Hb estimation-
• CYANMETHEMOGLOBIN METHOD- GOLD STANDARD
• ALKALI HEMATIN METHOD
• OXYHEMOGLOBIN METHOD
• AUTOMATED ANALYSER

• NORMAL- MALES:13-18gm%;
females:12-16gm%
INCREASED HEMOGLOBIN DECREASED HEMOGLOBIN
High altitude Anemias
Obstructive lung disease Parasitic infestations
Polycythemia- primary and secondary Pregnancy
Congenital heart disease Drugs
Smoking Lead poisoning
Iron deficiency
Kidney disease
 WBC PIPETTE
• IDENTIFICATION
• White bead-Bead helps in identification, aids in mixing, tells
whether the bulb is dry or not(bead sticks to bulb in case of wet
bulb)
• Markings- 0.5, 1, 11

Dilution factor-20
Diluting fluid- TURK’S FLUID
• USES
• WBC count(4000-11000/Cumm in adults)
• Absolute Eosinophil count
• Platelet count
• Sperm count
• Cell count in CSF/Pleural/Ascitic tap
• RBC count when patient have severe anemia
RBC Pipette
• Red bead
• Markings – 0.5,1 and 101
• Dilution factor-200
• RBC diluting fluid- HAYEM’S
FLUID , DACIE’S FLUID

• USES
• RBC count(NORMAL :
3.8-5million/cumm females
and 5-6million/cumm males)
• High WBC count e.g. Acute
leukemias
• Sperm count
MICROPIPETTES
 IMPROVED NEUBAUER
CHAMBER
• Earlier Neubauer’s chamber-
16 squares in all the 9 big
squares, improved chamber
has 25 squares in the
central square
• Area 3mmX3mm
• Area of each small square –
1mmX1mm
• 16 small squares are
separated by bold lines
• Corner squares are divided
into 16 squares
• After charging depth of
chamber 0.1mm
• USE
• CORNER SQUARE- TLC, SPERM COUNT, AEC
• 5 SMALL SQUARES IN THE CENTRAL SQUARE-RBC COUNT
• CENTRAL SQUARE- PLATELET COUNT
VACUTAINERS/VACUUM
TUBES
1. EDTA
• Does not affect the size and
number of cells
• salts available-
• Disodium(Na2)

• Tripotassium(K3), dipotassium(K2)

• K3 saltmore soluble than

sodium salts
USES
• Standard haematology anticoagulant- used for
• Hb
• RBC Count
• WBC count
• platelet count
• red cell indices
• PCV
• reticulocyte count
• HbA1C, Hb electrophoresis
• Malarial Parasite by card
• Mechanism of action: chelates/binds
calcium ions which are required for coagulation
• Concentration- 1.5 + 0.25mg/ml of blood

• If EDTA is used in excess, it causes shrinkage of

RBC,WBC, swelling of platelets
 2. TRISODIUM CITRATE

• Mechanism- combines with calcium

and removes it
• Used for
• coagulation studies in a
ratio of 1:9(BLUE CAP;
3.2%w/v)
• ESR studies in a ratio of
1:4(BLACK CAP-3.8%w/v)
COAGULATION STUDIES
• PROTHROMBIN TIME
• ACTIVATED PARTIAL THROMBOPLASTIN TIME
• THROMBIN TIME
• D DIMER
• FIBRINOGEN LEVELS
• FACTOR ASSAYS
3. HEPARIN
• Natural anticoagulant. Sodium/lithium salt
of heparin used
• Inactivates thrombin
• Preserves red cell size
• Concentration- 10-20 IU/ml
• E.g. osmotic fragility studies, G6PD
studies, flow cytometry
• Unsatisfactory for WBC and platelet
counts-clumping
• gives a faint blue background color after
staining
OTHER VACUTAINERS
• GREY CAP- FLUORIDE AS AN
ADDITIVE. Anticoagulant is EDTA
• USED for blood glucose
estimation
• Fluoride inhibits enzymes of
glucose metabolism e.g.
ENOLASE
• YELLOW CAP and RED CAP VACUTAINERS- For serum
studies
• CONTAIN NO ANTICOAGULANT
• E.G. Liver function tests, renal function tests, lipid profile,
serum electrolytes, thyroid function tests

• GOLD CAP VACUTAINER/ SERUM SEPARATOR TUBE- contains

Acrylic gel(polymer) helps in separating clot from serum
• Also the walls are silica coated, which act as a clot activator
NEEDLES
BONE MARROW
ASPIRATION
NEEDLES
• SALAH NEEDLE( “S for screw”)
• Parts- Guard(screw) , stylet and
needle

• KLIMA NEEDLE
• Needle, stylet SALAH NEEDLE

KLIMA NEEDLE
• SITES FOR BMA**
• PSIS- [Link] site is posterior superior iliac spine
• ASIS
• Sternum- in fatty patients
• Iliac crest
• Spinous process of vertebrae
• INFANT- Upper end of tibia
 INDICATIONS OF BONE
MARROW
• RBC disorders- Megaloblastic anemia, pure red cell aplasia,
Pancytopenia
• WBC disorders- Subleukemic/aleukemic leukemia, Acute Leukemias
• MEGAKARYOCYTIC DISORDERS- ITP, Undiagnosed thrombocytopenia
• Myeloproliferative neoplasms- Polycythemia vera, CML, Essential
thrombocythemia
• Perls prussian blue to assess Iron stores, demonstrate ring
sideroblasts
• Storage disorders- Gaucher’s disease, Niemann Pick’s disease
• Parasitic infections- Malaria, leishmania
• Metastasis, Granulomas- BM Biopsy is Superior
• THERAPEUTIC INDICATION- BONE MARROW TRANSPLANTATION
 STYLE
T

NEEDLE

Jamshidi trephine needle for bone

marrow biopsy.
BM BIOPSY
• In case of “DRY TAP” e.g. in aplastic anemia, myelofibrosis
• To diagnose aplastic anemia, hypoplastic bone marrows
• Focal pathologies- e.g. Granulomas, Metastasis
• To assess the topography, cellularity, architecture of bone
marrow

• ANOTHER NEEDLES FOR BM BIOPSIES- ISLAM NEEDLE, OSGOOD

NEEDLE
• C/I FOR BMA and BMB- Bleeding disorder, skin infection at local
site, Bone disease/Bone deformity at the site
LUMBAR PUNCTURE
NEEDLE
• USED FOR SAMPLING CSF

(NORMAL VALUES
VOL- 100-150 ml
Opening pressure- 60-180mmHg
Normal counts
Adults- 0-5/cumm
Infants- 0-30/cumm
Glucose- 45-80mg/dl
Proteins-15-45mg/dl) SITES-
ADULT- L3/L4
CHILDREN- L4/5
 LUMBAR PUNCTURE
DIAGNOSTIC THERAPEUTIC CONTRAINDICATIONS COMPLICATIONS

Meningitis- Viral, Spinal anaesthesia Marked increase in POST LP HEADACHE

Pyogenic and CSF pressure
tubercular
Chemotherapeutic
Metastasis/ Patient with brain INTRODUCTION OF
drugs
lymphoma cells tumor INFECTION
Encephalitis Any local infective Brainstem
lesion HERNIATION IN C/O
INCREASED ICP
Sub arachnoid
haemorrhage
Spinal cord tumor
To inject
radioopaque dye
for myelography
 CSF findings in meningitis
FINDINGS PYOGENIC M. ASEPTIC M. TUBERCULAR M.

Naked appearance Cloudy/purulent clear Cobweb coagulum

Pressure Elevated Elevated Elevated

Predominant cells Neutrophils Lymphocytes Lymphocytes

Protein level Increased Normal Increased

Sugar content decreased Normal decreased

Bacteriological Organism Sterile AFB Staining

study demonstrable-
Gram staining
INSTRUMENTS FOR ESR
• METHODS
• Westergren,s method
• Wintrobe’s method

• STAGES OF SEDIMENTATION**
1. ROULEAX FORMATION
2. FORMATION OF FINE THREADS
3. RAPID FALL
4. PACKING PHASE
WESTERGREN’S
PIPETTE
• Both sides open
• 30cms in length
• 2.5mm in dm
• Calibration 0-200 at 1mm interval
• Markings top to bottom
• ADVANTAGE- MORE ACCURATE
METHOD than wintrobe’s method
Westergren’s pipette in westergren's stand
• PRECAUTIONS-
1. Tube should be VERTICAL. An angle of 3 degrees increases
ESR by 30%
2. SAMPLE Should be taken in 3.8%w/v Trisodium citrate in a
ratio 1:4(ANTICOAGULANT:BLOOD)

INCREASED ESR DECREASED ESR

Chronic infection [Link] POLYCYTHEMIA VERA

Chronic inflammatory disease e.g. Sickle cell anemia

Rheumatic fever, Rheumatoid arthritis

Hypergammaglobinemias- Multiple Hyperfibrinogenemia

myeloma
WINTROBE’S TUBE
Closed at one end
EDTA Blood is used
Length 110mm
Internal dm- 2.5mm
Calibration- 1mm
interval to 100mm

MARKINGS
From top
0,10,20…….100 for ESR
From top- 100, 90,
80…….0 for PCV
ESR NORMAL RANGE
WINTROBE’S METHOD WESTERGREN’S
METHOD
(mm in 1st hour) (mm in 1st hour)

MALE 0-7 0-10

FEMALE 0-14 0-20

 NOTE
• WINTROBE’S TUBE CAN BE USED TO
MEASURE PCV ALSO
• READING TAKEN FROM BELOW
UPWARDS
• USES OF COLOUR OF PLASMA
• NORMAL RANGE
• MALE-40-55%
• FEMALE 38-45%
• INFANTS:45-60%
Variation in Haematocrit:
Increased – Polycythemia vera.
Decreased – Anemias Straw coloured -normal
INFO FROM COLOUR OF PLASMA Deep yellow- Jaundice
Turbid- Hyperlipidemia
Red - hemoglobinemia
• BUFFY COAT USE

• LE cell
phenomenon
• Detection of
Malarial
parasite(QBC
method)
OTHER METHOD FOR PCV/HAEMATOCRIT

• MICROHAEMATOCRIT METHOD- Uses less amount of blood

sample
URINOMETER
• INDICATION- TO MEASURE SPECIFIC GRAVITY OF URINE
• Calibrated at 20 degree celsius
• OTHER METHODS- REFRACTOMETER, DIPSTICK METHOD
• PRINCIPLE- urinometer floats higher in urine than
water(denser than water)

• NORMAL RANGE-
• RANDOM SAMPLE- 1.003-1.030
• 24 HR URINE SAMPLE- 1.015-1.025
• ISOSTHENURIA- Low and fixed specific gravity of 1.010
([Link] FAILURE- Sp gr same as that of plasma)
PRECAUTIONS &
CORRECTIONS
• Urinometer calibrated at 20 degrees. For every 3 degree rise
in temperature 0.001 is added and for every 3 degree fall in
temperature 0.001 is subtracted
• Correction for protein and glucose
• ACTUAL= Observed-0.003 for each 1g/dl protein
• Actual= Observed-0.004 for each 1g/dl glucose

• URINOMETER SHOULD NOT TOUCH THE WALL OF THE

CONTAINER
• READING IS TAKEN AT LOWER MENISCUS
• Fill the glass cylinder with urine up to 2/3rds. At least 15 ml of
urine is required
SPECIFIC GRAVITY
INCREASED DECREASED
Dehydration Excessive fluid intake

Decreased fluid intake Diabetes insipidus

Diarrhoea End stage kidney disease(Low and

Vomiting Fixed)

Fever
Excessive sweating
Diabetes mellitus
Albuminuria
ESBACH’S ALBUMINOMETER
• Used for quantitative estimation of protein in 24 hr urine
sample
• Esbach’s tube – two markings U and R
• ESBACH’S REAGENT- Citric acid(dissolves phosphate) and
picric acid(precipitates albumin)
• Urine filled upto U mark,Esbach’s reagent upto R mark

• REFERENCE VALUES
• Normal protein excretion-Upto 150mg/24hrs
• Microalbuminuria: 30-300mg/24 hrs
• Nephrotic range proteinuria>3.5gms/24hrs
CAUSES OF PROTEINURIA**
• Muscular exertion
• High fever
• HTN
• Orthostatic albuminuria
• Kidney disease-
• nephrotic syndrome
• Acute glomerulonephritis
• Renal Tuberculosis
• Renal cell carcinoma
• Renal vein thrombosis
PAP SMEAR KIT

ENDOCERVICAL BRUSH

AYER’S SPATULA
 NEEDLE WITH
SYRINGE
USES
• To withdraw blood
• To perform fine needle aspiration
cytology
• In bone marrow aspiration
FNAC HANDLE/
FRANZEN,S HANDLE/
PLUNGER

• TO PERFORM FINE
NEEDLE
ASPIRATION
CYTOLOGY
PROCEDURE
REFERENCES
• Practical Pathology, [Link] Singh
• Google images
THANK
YOU!!!
THANK
YOU!!!