D irect M icroscopic S omatic C ell C ount 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

R ules 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

R ules contd 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.

R ules contd 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.

Form FDA 2400d (6/05) DMSCC 25.g. Identifying and counting somatic cells. 1.Cells possess a nucleus stained dark blue (bovine) or blue- green (caprine). 2.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. 3.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. 4.Count cells touching only the top or bottom half of the strip. 5.IF IN DOUBT, DO NOT COUNT.

A B C 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 8 microns D

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

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

AB 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

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

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

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

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

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

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

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

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

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

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. C is a typical multi-lobed cell. (2 cells) A B C 14

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) A B D C 15

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

D A B C E F 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

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

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

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

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

B C A 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

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

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

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

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

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

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

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

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

F inal C ount The total count for 30 slides of one strip count = 88 cells