Wskazania dotyczące organizacji laboratorium, wykonywania rozmazów i skriningu.

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Etapy pracy w laboratorium cytologicznym:

• Laboratorium otrzymuje preparat i za??cznik do badania.
• Wprowadzenie referencyjnego numeru laboratorium.
• Wprowadzenie danych identyfikacyjnych pacjentki i numeru rozmazu do bazy komputerowej.
• Barwienie, nakrywanie i oznaczenie rozmazu.
• Pierwotny skrining.
• Ocena wszystkich rozmazów przez drugiego skrinera, konsultacja rozmazów nieprawid?owych z patologiem.
• Napisanie wyniku badania i wys?anie go do tego, kto rozmaz pobra?.
• Poinformowanie pacjentki o wyniku.
• Upewnienie si?, ?e odpowiednie dzia?ania zosta?y podj?te w przypadku nieprawid?owego wyniku.

 

Przyj?cie rozmazów do laboratorium

1. Sprawdzic czy rozmaz i za??cznik nale?? do tej samej kobiety. Wpisac ten sam numer na rozmazie i na za??czniku (lub kod kreskowy).
2. Zabarwic rozmaz metod? Papanicolaou.
3. Nakryc rozmaz szkie?kiem nakrywkowym w DPX (optymalne rozmiary szkie?ka nakrywkowego 22x50mm).
4. Medium u?yte do nakrycia szkie?ka powinno wyschn?c zanim wraz z za??cznikiem pow?druje do pierwotnego skrinera.

Barwienie metod? Papanicolaou.

Papanicolaou opracowa? stosowan? do dzisiaj z niewielkimi modyfikacjami trichromow? metod? barwienia rozmazów Papanicolaou G.: A new procedure for staining vaginal smears. Science 95,438,1942). Optymalne wyniki uzyskuje si? je?eli rozmazy nie wysychaj? ani na chwil? przed utrwaleniem i w czasie barwienia.

Barwienie Papanicolaou

#	Procedure	Time	Comment
1	Remove polyethylene glycol carbowax fixative In 50% alcohol	2 min’s	Failure to remove carbowax will result in artifact which obscure the cells & is difficult to remove
2	Rinse in water	1 min	Ensure re hydration is complete
3	Stain in Harris Haematoxylin	5 min’s	Staining time will depend on formula of haematoxylin
4	Rinse in water	2 min’s	Unbound haematoxylin is removed
5	Differentiate in 0.5% aqueous hydrochloric acid	10-20 sec’s	_
6	Rinse in tap water	2 min’s	_
7	Blue in Scott’s tap water substitute	1 min	he brown colour of acid haematin is changed to blue/ black by weak alkaline solution 8
8	Rinse in water	2 min’s	Wash thoroughly to remove alkaline salts
9	Dehydrate 70%alcohol	2 min’s	Dehydrate through graded alcohols
10	Dehydrate  95%alcohol	2 min’s
11	Dehydrate 95%alcohol	2 min’s
12	Stain in Orange G6 (OG6)	2 min’s
13	Rinse in 95% alcohol	2 min’s
14	Rinse in 95% alcohol	2 min’s
15	Rinse in 95% alcohol	2 min’s
16	Stain in Gills EA30 solution	3 min’s
17	Rinse in 95%  alcohol  x3	1 min
18	Clear in xylene x3 and mount in DPX	1 min	Leave in xylene until ready to mount in DPX

Resulting stain:  

• Nuclei- blue black
• Cytoplasm- non keratinised blue and effete red
• blood cells- green
• Cytoplasm (keratinised)- pink
• Red cells- orange

The end result should retain the transparent quality of the cytoplasm and the chromatin structure of the nuclei  should be clearly defined.

Comment on the Papanicolaou staining method:

The Papanicolaou stain is a polychrome staining method which comprises a nuclear stain (haematoxylin) and two counterstains (Orange G and EA dyes). Hydration of the fixed smear is required for the cells to take up the haematoxylin whereas dehydration prepares the smear for the counterstains. Modification of the stain is common as each laboratory prefers its own colour balance. Adjustments are made by altering the length of time in haematoxylin and EA dye. Other factors which may affect the colour balance are chemical content of the tap water, temperature, pH of specimen and number of slides per batch of stain.   Providing that the nuclear detail is clearly defined and the transparency of the cytoplasm is maintained, interlaboratory variation of the colour balance is acceptable. However each laboratory should standardise its procedure so the results are reproducible.

The Papanicolaou staining method may progressive or regressive.

In the progressive method the intensity of nuclear staining is controlled by immersion of the slide in a “blueing” agent after the nucleus has been stained to the required intensity with haematoxylin. The blueing agents most commonly used are Scott’s tap water substitute (pH 8.02), ammonium hydroxide and lithium carbonate. (Tap water can be used if the pH is suitable). Progressive staining tints the cytoplasm very lightly.

In regressive staining, the nucleus is deliberately overstained with a non-acidified haematoxylin. The excess stain is removed with dilute hydrochloric acid solution. The decolourising acid is then removed by immersing the slide in running tap water. Timing is important in the regressive method as the outcome may be a hypochromatic nucleus rather than a hyperchromatic nucleus. The cytoplasm is also totally decolourised by the acid solution. Harris’s haematoxylin is usually combined with the regressive staining method. Gill’s or Mayer’s haematoxylin is usually used for the progressive staining method. It is important to remember that the pH of the solution is more important than whether the haematoxolin is used progressively or regressively.

Orange G is an acidic dye which stains basic proteins such a prekeratin a pink colour. Orange G also has a strong affinity for keratin which stains bright orange. Thus it is an important marker of abnormal keratinisation of the cervical and vaginal epithelium such as occurs in prolapse and wart virus infection. It is also a sensitive marker of well differentiated squamous carcinoma of the cervix. It is of utmost importnace that the rinsing baths are clean and the staining solutions are changed frequently otherwise both nuclear and cytoplasmic staining will be defective.

EA is a polychromatic stain which is a combination of light green, SF yellow and eosin Y. It stains the cytoplasm of metabolically active cells (such as parabasal cells intermediate cells leucocytes and histiocytes as well as cancer cells) a light green colour.
However, the colour balance of a Papanicolaou stained slide depends not only on the staining method used but also the commercial source of the stains and the pH of the cell sample. In consequence the Papanicolaou stain cannot be considered to be an exact stoichiometric stain although the correlation between Pap stained and Feulgen stained material is reportedly very high.

For further information about the Papanicolaou stain and general cytopreparatory techniques please consult Chapter 34 , Cytopreparatory Techniques by CM Keebler in Comprehensive Cytopathology (1996) editor M Bibbo pub Saunders.   

Screening and reporting

• A protocol for screening should be agreed with the laboratory manager and quality control measures and appropriate staff/ workload ratios should be in place.
• In order to develop expertise it is essential that cytotechnologists see a wide range of cases. Laboratories should process at least 15,000 cervical smears per year and each screener may be expected to screen 50 -80 smears per day  with breaks at hourly intervals. He/she should have his/her own workstation and binocular microscope.
• The prepared slides should  be read using  a 10x objective and 10x or 12x eye pieces. The same objective should be used to screen the whole slide. The screener should progress field by field across the slide until the whole area under the coverslip has been inspected. It is usual to start screening at one corner of the slide and work in a step wise manner until the whole area under the coverslip has been examined as shown below.

 

• It has been estimated that, using low power objectives, a screener will have examined  250-300 fields of view  during the process of screening a single slide. The average time taken to complete this process is 6-10 minutes (range 5 -20 minutes).
• To mark cells of interest, a 4x objective should be used. A  protocol for marking cells should be in place  in the laboratory.
• To inspect areas of special interest  a 20x or 40x objective should be used.
• The cytology request form and cervical screening history should be provided with  each smear and  should be reviewed by the cytologist before examining the smear.

Terminologia i zasady formu?owania wyników bada? cytologicznych szyjki macicy

Wynik badania cytologicznego powinien by? dok?adny i konkretny tak aby by? ?atwo zrozumia?y dla pobieraj?cego wymaz, i pracowników medycznych, technicznych i administracyjnych zaanga?owanych w program skriningowy.
Terminologia musi przekazywac istotn? klinicznie informacj?. Powszechnie si? uwa?a, ?e mo?na to osi?gn?c najlepiej pisz?c wynik w postaci d?u?szego wyja?niaj?cego tekstu poniewa? wyniki formu?owane skrótowo mog? prowadzi? do b??dnych interpretacji. Z tego powodu ,zdecydowanie odradza si? stosowania klasyfikacji numerycznych z klasyfikacj? Papanicolaou w??cznie.

Wynik badania cytologicznego sk?ada sie z pi?ciu cz??ci:

1. Rodzaj preparatu, np. wymaz z szyjki, wymaz szczoteczkowy z kana?u szyjki lub LBC.
2. Komentarz na temat jako?ci rozmazu tzn. czy jest odpowiedni do oceny.
3. Krótki opis stwierdzonych zmian cytologicznych w sposób powszechnie stosowany i zrozumia?y.
4. Je?eli rozmaz jest nieprawid?owy nale?y podac jakim zmianom patologicznym stwierdzone cytologiczne zmiany powinny odpowiadac.
5. Sugestie dotycz?ce dalszego post?powania (mog? ale nie musz? by? wyra?one).

• Przed przygotowaniem wyniku badania cytologicznego cytolog powinien si? zapoznac z odpowiednimi danymi klinicznymi.
• Rekomenduje si? z naciskiem aby wyniki bada? wszystkich nieprawid?owych rozmazów by?y formu?owane przez patologa lub praktyka z odpowiednimi kwalifikacjami, który zosta? specjalnie przeszkolony w patologii ginekologicznej.