D irect M icroscopic S omatic C ell C ount Guidelines

1. Direct Microscopic Somatic Cell Count Guidelines Prepared By: Laboratory Development Section Division of Microbiology Bureau of Foods Food and Drug Administration November 1972 Adapted to Slides By: DHHS,PHS,FDA,CFSAN,OC,DCP Laboratory Quality Assurance Branch January 2003

2. Rules for identifying and counting somatic cells • Leukocytes have a nucleus • Nuclear mass composition • Recognizable form • One or more lobes • Lobes or units are bridged by nuclear material • Some cells are granular in appearance • Some distortion is expected because of treatment • Nuclear mass stains dark blue or blue-green • Count nuclear mass that bears resemblance to a typical nucleus

3. Rules cont’d • Cytoplasm normally surrounds the nucleus. • Stains light blue, • Not stained and appears as a clear zone, or • Disintegrated and not present. • Do not count cells without a stained nuclear mass (ghost cell) • Cell size. • Nuclear mass of a countable cell is ≥ 8 microns. • Fragments of a countable cell with at least 50% of the nuclear mass visible and ≥ 4 microns.

4. Rules cont’d • Clump of cells • Nuclear mass must be clearly delineated to count individual cell(s). • If no clear delineation, count clump as one cell. • Field boundaries • Do not over extend the field or strip boundaries • Count cells touching top or bottom edge of strip, not both.

5. Form FDA 2400d (6/05)DMSCC 25.g. Identifying and counting somatic cells. • Cells possess a nucleus stained dark blue (bovine) or blue-green (caprine). • Cells generally 8 microns or larger (bovine, caprine may be smaller); do not count cells less than 4 microns; fragments counted only if more than 50% of nuclear material is visible. • Cluster of cells counted as one unless nuclear unit(s) are clearly separated; focus up and down to ensure that there are no bridges connecting nuclear masses. • Count cells touching only the top or bottom half of the strip. • IF IN DOUBT, DO NOT COUNT.

6. 8 microns C A B 8 microns D A, B and C are countable cells. D is not a countable cell since it is less than 8 microns in diameter. Circled items are bacteria or artifacts that bear no resemblance to a typical nucleus and not counted. (3 cells) 1

7. A B A and B are countable cells. (2 cells) 2

8. A A is a countable cell. (1 cell) 3

9. A B A and B are countable cells. Both are surrounded by disintegrating cytoplasm, however the nuclear material remains intact and each resembles a typical nucleus. (2 cells) 4

10. E A B D C A – D are typical cells (multi-lobed nuclei). E is cellular debris and not counted. (4 cells) 5

11. Nuclear bridge A One cell with nuclear lobes connected by a nuclear bridge. (1 cell) 6

12. C A B D B and D are typical cells. (2 cells) A and C are cytoplasmic debris and not counted. 7

13. A B B is a countable cell. A is a ghost cell (no nuclear material) and not counted. (1 cell) 8

14. A One cell with a disintegrating nucleus, however greater than 50% of the multi-lobed nuclear mass is visible. (1 cell) 9

15. B E A C D Nuclear Bridge A-E are typical cells. (5 cells) 10

16. A B A is a typical cell with a multi-lobed nucleus. B is a typical monocyte. Both are counted. (2 cells) 11

17. A A is a single cell with cytoplasm. (1 cell) 12

18. B F C A D E Nuclear bridges All are cells. B and E are bi-lobed with nuclear bridges. (6 cells) 13

19. C A B A is less than 8 microns in diameter and not counted. B has a disintegrating nucleus with greater than 50% of the nuclear mass visible and is counted. 14 C is a typical multi-lobed cell. (2 cells)

20. D B C A A, C, and D are typical cells. Cell D is touching the top edge of the field and not counted. B is cytoplasmic debris (less than 50% of nucleus present) and not counted. (2 cells) 15

21. H B E C D G F A Nuclear bridge? A-G are typical cells. Cell B is not counted as it touches the top edge of the field. (7 cells) 16

22. F C B D E A A, B, C, and E are typical cells. D is a cell with a large amount of disintegrating cytoplasm. F is debris, not a cell. (5 cells) 17

23. A B C A-C are typical cells. Blue-circled item is a bacteria or artifact that bears no resemblance to a typical nucleus and not counted as a cell. (3 cells) 18

24. C F A D E B Cell A appears to be >8 microns. Cell C is not counted as it touches the top edge. B, D, E, and F are typical, countable cells. (5 cells) 19

25. A B A and B are debris. (0 cells) 20

26. A B C A is a typical cell. B and C are cytoplasmic debris. (1 cell) 21

27. A B C D A and D are typical cells. Cell B is a fragment greater than 50% of original size. Cell C has disintegrating nuclear material greater than 50% of original composition. (4 cells) 22

28. A B C D A and B are typical cells. C and D are cells showing early degeneration. (4 cells) 23

29. A A is a typical cell. (1 cell) 24

30. A E B C D A – E is a clump of typical cells. (5 cells) 25

31. A J D I F E G H C B A, B, and I are bi-lobed cells. C is tri-lobed. D contains 4 nuclear masses. E, F, G, and H are mononuclear cells. J is a disintegrating cell. (10 cells) 26

32. A B C D Nuclear Bridges? A, B, C, and D are typical cells. (4 cells) 27

33. A B A is a typical cell showing cytoplasmic degeneration. B is cellular debris. (1 cell) 28

34. A C B A and B are typical cells, but A is touching the top edge and not counted. C is a ghost cell. (1 cells) 29

35. A B A is a disintegrating cell. B is typical. (2 cells) 30

36. Final Count • The total count for 30 slides of one strip count = 88 cells