This document discusses cytopreparatory techniques for cytology samples. It describes the different types of cytology samples like exfoliative cytology, aspiration cytology, and body fluids. The key steps in cytopreparatory techniques are outlined as evaluation of specimens, preparation of smears, fixation, and staining. Factors that can affect optimal cytological preparation like quality of specimen, fixative used, and stains are also summarized. Different fixation techniques including dry, wet, liquid-based, and lysing fixation for bloody samples are explained.

1. Duhok University
Faculty of medical science
School of health science
Cytology lecture series
Dr. Azad Mustafa Ahmed
Lecturer, FIBMS (Path)
University of Duhok, faculty of medical science

2. Lecture 2
Cytopreparatory Techniques

3. Cytopathology
is a branch of pathology that study and
diagnose diseases by evaluation of the
cellular changes that happen to the cells.

4. Types of cytology samples
Exfoliative
cytology
Aspiration cytology
(Fine Needle
Aspiration Cytology
(FNAC)
Body fluids

5. Exfoliative cytology
• It is the study of cells that have been shed or removed from
the epithelial surface of various organs.
wash smear
scraping brushing

6. Bronchial wash

7. Brushing

8. brush

9. Smear or scrapping

10. Aspiration cytology (Fine Needle Aspiration Cytology (FNAC)
• This is a technique used to obtain material from organs or
lesions by needle aspiration. It is valuable in diagnosis of
lesions of the breast, thyroid, lymph nodes, liver, lung, soft
tissue …etc.

16. Body fluids
• Body fluids like Urine, Pleural fluid, Pericardial fluid,
Cerebrospinal fluid, Synovial fluid and Ascitic fluid can be
studied for cytopathology.

17. Pleural fluid

18. Pericardial fluid

19. Cerebrospinal fluid CSF,

20. Synovial fluid

21. Ascitic fluid

22. Advantage of cytopathology:
1. a non-invasive procedure,
2. helps in faster reporting and guide the clinician
3. is relatively inexpensive
4. Has high population acceptance
5. Facilitates cancer screening.

23. Factors affecting optimal cytological preparation
• quality of the specimen
– too bloody, too thick, or has been left too long before being
taken to the laboratory, the results will be less than optimal

24. Factors affecting optimal cytological preparation
• fixative chosen to preserve the cellular details:
– If fixative not correct for the specimen or the fixative is not
applied quickly, the results will again be less than optimal.

25. Factors affecting optimal cytological preparation
• Stain used to render cells viewable:
– If the stains are old, not properly prepared, or not
optimized for time, again the results will not be optimal.

26. Steps in Cytopreparatory Techniques:
1. Evaluation of specimens
2. Preparation of smears
3. Fixation
4. Staining and coverslipping
Evaluation Smear fixation staining

27. Evaluation of specimen:
• The specimens should be evaluated in order to determine the
pathway to adequate cellular preparation e.g.
– Bloody samples may need additional treatment with a lytic
agent such as "cytoLyt" to lyse the RBCs.

28. Evaluation of specimen:
• Mucoid specimens need the addition of a mucolytic agent such
as "Mucolexx" to thin and break up the mucus so it can be
processed further

29. Gross examination
• Determine the volume in ml
• Color: yellow, milky or brown.
• Appearance: clear, turbid or hemorrhagic

30. Preparation of smears

31. Preparation of smears
• It means spread of obtained material on a glass slide.
• The smears are usually made directly from the aspirated fluid,
as rapid as possible to prevent dry artifacts on the cells.

32. Preparation of smears
• If delay is anticipated, the fluids kept at the refrigerator for 24-
48 hours or by adding ethanol as a fixative to the fluid (2:1)
which can keep the sample for 7 days or more

33. Preparation of smears
• Centrifuge the fluid:
– If too much fluid is obtained, it needs to be centrifuged for 5
minutes, and then the sediment is used to make a smear
because it is more cellular.
– If little amount of fluid is aspirated (few drops), or if the
fluid is thick, the centrifuge doesn’t required.

34. Preparation of smears
• Cytocentrifuge: It is a special machine that performs a
centrifuge and collection of sediment on the center of the
slides. This procedure is useful for hypocellular specimen like
urine for cytology.

35. Preparation of smears
• Liquid based cytology LBC: These specimens are received in
special vial that contain special solutions to preserve the cells
inside and then the slide is made by special automated
machines. The basic principles are the vaccum and special
filtering with collection of diagnostic material at the center of
the slides.

38. Cytology smear preparation
Cytology

39. Fixation
The purpose of cytologic fixatives is to maintain, as closely as
possible, the cytomorphologic characteristics and diagnostically
essential cytochemical elements of the cell.

40. An appropriate fixative for cytodiagnostic
purposes should perform the following functions:
1. Penetrate cells rapidly
2. Minimize cell shrinkage
3. Maintain morphologic integrity
4. Deactivate autolytic enzymes
5. Replace cellular water
6. Facilitate diffusion of dyes across cell boundaries
7. Help cells adhere to a glass surface
8. Provide consistent results over time
9. Produce a permanent cell record
10. Stop cellular and microbial growth (antimicrobial).

41. Types of fixative:
• Dry fixation:
• Wet fixation:
• Liquid-based Fixation:
• Lysing Fixation for Bloody Samples:

42. Dry fixation:
• The slide is dry by air quickly after the material is spread on the
slide.
• Followed by hematological stains like Wright–Giemsa, Diff-
Quik, or May–Grünwald–Giemsa staining procedures.

43. Wet fixation:
1. Ethanol 95% is the best wet fixative
2. Wet Fixation with subsequent Air Drying: used for transfer
slides to the laboratory
3. Spray Fixation: contain alcohol with wax for better
preservation for transportation

44. Liquid-based Fixation
• the sample is collected to vial contains fixative solutions mostly
methanol. This type of fixative is suitable for transport of
specimen to other place and can be stored up to 7 days or
more.

45. Lysing Fixation for Bloody Samples:
1. Carnoy’s solution: absolute ethanol, chloroform glacial acetic
acid (6 : 3 : 1)
2. One drop of concentrated hydrochloric acid per 500 mL of 95%
ethanol
3. Ready used preparation: CytoLyt, CytoRich Red etc.

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