• Body fluid cytology is vital in diagnosis of various neoplastic and non neoplastic lesions and conventional Pap stain is the staining method of choice for the same. • MUFP is a quick and cheap staining technique which gives good interpretation of cytological features with easily available reagents. • Preservation of cell morphology and nuclear staining are superior with conventional Papanicolaou technique whereas cytoplasmic staining is comparable with conventional pap and MUFP techniques. • Though background of stained smears was slightly better with conventional Pap staining; MUFP was superior in case of hemorrhagic samples.

1. COMPARISON OF CONVENTIONAL PAPANICOLAOU
STAIN WITH MODIFIED ULTRAFAST PAPANICOLAOU
STAIN IN BODY FLUID CYTOLOGY
KHMD.U4MLT13013

2. INTRODUCTION
 Papanicolaou (Pap) staining method has revolutionized cytology and
remains the gold standard in cytology staining.
 To overcome limitations of routine Pap staining several modifications
were proposed.
 Yang and Alverez in 1995 suggested the use of Ultrafast Papanicolaou
stain.
 This technique was further modified as many of the reagents were not easily
available in our country - Modified Ultra Fast Papanicolaou stain
(MUFP).

3. AIM AND OBJECTIVES
 To compare the efficacy of modified ultrafast Papanicolaou staining and
conventional Papanicolaou staining methods in body fluid cytology.
 To assess whether modified ultrafast Papanicolaou stain can replace
conventional Papanicolaou staining for various practical purposes in body
fluid cytology.

4. MATERIALS AND METHODS
 The study included 65 body fluid samples including 29 ascitic fluid
specimens, 16 pleural fluid specimens, 19 peritoneal fluid specimens,
and 1 pericardial fluid specimen received in our cytology laboratory
during the period from January 1st to February 28th for routine
cytologic evaluation.

5. STAINING
The samples were processed, slides prepared and stained by the
following methods
 CONVENTIONAL PAP STAINING
 MODIFIED ULTRAFAST PAP STAINING
 For Pap Staining, the prepared smear was fixed in 95 %
isopropyl alcohol or 95 % ethyl alcohol without any delay
 For MUFP, the smear was air dried, placed in normal saline for
30 seconds and fixed in alcoholic formalin for 10 seconds

6. STAINING PROCEDURE
MODIFIED ULTRAFAST PAP STAINING
 Tap water – 6 slow dips
 Harris Hematoxylin – 30 seconds
 Tap water – 6 slow dips
 95 % Isopropyl alcohol – 6 slow dips
 EA 36 – 15 seconds
 95 % isopropyl alcohol – 6 slow dips
 100 % isopropyl alcohol – 6 slow dips
 Xylene – 10 dips
 After clearing, mount using DPX
80 % Ethyl alcohol (10 dips
)
Dips
70 % Ethyl Alcohol 10 dips
50 % Ethyl Alcohol 10 dips
Distilled Water 2 rinses
Harris Hematoxylin 6
minutes
Distilled water 2
rinses
0.5 % Aqueous HCl two dips
Running tap water 10 dips
Lithium carbonate solution 1 dip
Distilled water 10 dips
70 % Ethyl Alcohol
10 dips
95 % Ethyl Alcohol (10
dips)
Dips
Orange G 2 minutes
95 % Ethyl Alcohol 10 dips
95 % Ethyl Alcohol 10 dips
EA 36 10 dips
95 % Ethyl alcohol 10 dips
100 % Ethyl alcohol 10 dips
Xylene 10 dips
Xylene 10 dips
Xylene 10 dips
Mounting in DPX

7. EVALUATION OF STAINING QUALITY
 The stained smears were examined and compared for quality of staining
method employed in terms of fixation, nuclear staining, cytoplasmic
staining, artifacts and background of the smear.
 Each criterion was graded and scored individually as 0 , 1, 2 and 3.
FIXATION GRADE
No cells seen 0
Cell morphology is not
preserved well
1
Moderate preservation 2
Excellent preservation 3
NUCLEAR STAINING GRADE
No cells seen 0
Pale or dark 1
Good nuclear staining 2
Excellent nuclear
staining
3
CYTOPLASMIC
STAINING
GRADE
No cells seen 0
Poor cell differentiation 1
Moderate cell
differentiation
2
BACKGROUND GRADE
No cells 0
Dirty background 1
Obscured background 2
Clean background 3
ARTIFACTS / STAIN
DEPOSITS
GRADE
No cells 0
Marked artifacts/stain
deposits
1
Some artifacts/stain
deposits
2
No artifacts/stain
deposits
3

8. DISCUSSION
100
53.8
0
20
40
60
80
100
120
PAP MUFP
Percentage(%)
Grade 3
FIXATION GRADE- 3
MUFP
PAP
100
56.9
0
20
40
60
80
100
120
PAP MUFP
Percentage(%)
Grade 3
NUCLEAR STAIN GRADE- 3
MUFP
PAP

9. COMPARISON FOR CYTOPLASMIC STAINING IN PAP AND
MUFP STAINING METHOD
81.5 81.5
0
10
20
30
40
50
60
70
80
90
PAP MUFP
Percentage(%)
Grade 3
CYTOPLASMIC STAIN GRADE-
3
MUFP
PAP
18.4 18.4
0
2
4
6
8
10
12
14
16
18
20
PAP MUFPPercentage(%)
Grade 2
CYTOPLASMIC STAIN GRADE- 2
MUFP
PAP

10. COMPARISON OF BACKGROUND OF SMEAR IN PAPAND MUFP
STAINING
50.7
36.9
0
10
20
30
40
50
60
PAP MUFP
Percentage(%)
Grade 3
BACKGROUND GRADE- 3
MUFP
PAP
49.2
63
0
10
20
30
40
50
60
70
PAP MUFP
Percentage(%)
Grade 2
BACKGROUND GRADE- 2
MUFP
PAP

11. COMPARISON OFARTIFACTS OBTAINED IN PAPAND MUFP
STAINING METHOD
95.3
90.7
0
20
40
60
80
100
120
PAP MUFP
Percentage(%)
Grade 3
ARTIFACTS GRADE- 3
MUFP
PAP
4.61
9.23
0
1
2
3
4
5
6
7
8
9
10
PAP MUFP
Percentage(%)
Grade 2
ARTIFACTS GRADE- 2
MUFP
PAP

12. CONVENTIONAL PAP STAIN – ASCITIC FLUID
MODIFIED ULTRA FAST PAP STAIN - ASCITIC FLUIDIN HAEMORRHAGIC PLEURAL FLUID SAMPLE

13. CONCLUSION
 Body fluid cytology is vital in diagnosis of various neoplastic and non neoplastic
lesions and conventional Pap stain is the staining method of choice for the same.
 MUFP is a quick and cheap staining technique which gives good interpretation of
cytological features with easily available reagents.
 Preservation of cell morphology and nuclear staining are superior with
conventional Papanicolaou technique whereas cytoplasmic staining is comparable
with conventional pap and MUFP techniques.
 Though background of stained smears was slightly better with conventional Pap
staining; MUFP was superior in case of hemorrhagic samples.

14.  Hence, MUFP is a useful staining method for body fluid cytology for
all practical purposes though the quality of cell staining is slightly
inferior to conventional Pap.
 The lower cost and easy availability of reagents also make it a good
technique for rapid staining though it cannot replace conventional
Pap stain. Universal standardisation of MUFP stain needs to be done
to increase its utility.