Biological specimen evaluation methods using cytology and immunology
Information on cytokines and cytology obtained from a biological specimen are combined as a method of predicting the risk that dysplasia will progress to cancer. Methods are disclosed herein to augment the evaluation of biological samples from subjects being tested for cancer. In addition to the cell types that are traditionally considered in the morphology-based cytological screening of specimens, methods disclosed herein add evaluations of certain cell types and cytokines that are traditionally discounted or ignored during screening.
1. A method for estimating the risk of dysplasia progressing to a higher degree of dysplasia or to cancer in a subject, the method comprising:
(a) determining whether one or more anti-inflammatory cytokines are present in a cytological preparation of a biological sample comprising cells from the subject;
(b) morphologically classifying each cell in which it is determined that one or more anti-inflammatory cytokines is present, as an epithelial cell, a cell type of the innate immune system, or another type of cell;
(c) further morphologically classifying each epithelial cell in which one or more anti-inflammatory cytokines are present as non-dysplastic, dysplastic or cancerous;
(d) sub-classifying dysplastic epithelial cells according to the degree of dysplasia present and/or optionally sub-classifying cells of the innate immune system by cell type;
(e) estimating the risk of dysplasia progressing to a higher degree of dysplasia or to cancer based upon whether at least one anti-inflammatory cytokine is present in dysplastic cells and/or cells of the innate immune system according to:
where: WNL =non-dysplastic epithelial cell;
DYSP=dysplastic epithelial cell; and
IMM=cell of the immune system;
and the risk is inferred from the following table:
Cell
Cytokine
Cell
Cytokine
Class
Present
Class
Present
Risk of Progression
WNL
No
IMM
No
Normal State
WNL
No
IMM
Yes
Moderate Increased Risk
DYSP
No
IMM
No
Slightly Increased Risk
DYSP
No
IMM
Yes
Moderately Increased Risk
DYSP
Yes
IMM
Yes
Greatly Increased Risk
wherein the presence of anti-inflammatory cytokines is determined using an immunofluorescence method comprising an unlabeled primary antibody and two identical secondary antibodies, wherein each antibody is labeled with a different fluorophore.
2. A method for the immunological determination of the spatial distribution of an analyte in a cytological or histological specimen, the method comprising:
a. contacting the specimen with an unlabeled primary antibody;
b. contacting the specimen with a reagent comprising two identical secondary antibodies wherein each antibody is labeled with a different fluorophore, and wherein the labeled secondary antibodies are bound to the specimen;
c. determining the locations on the specimen where the two labeled secondary antibodies are bound;
d. spatially correlating the locations on the specimen at which the bound first and second labeled secondary antibodies are present, wherein the analyte is inferred present at the locations where both the first and second labeled secondary antibodies are present, and the analyte is inferred to be absent where neither labeled secondary antibody is present, only one labeled secondary antibody is present, or both labeled secondary antibodies are present with low correlation,
wherein the cells in the specimen are contacted with a fluorescent or fluorogenic morphological stain and identified and/or classified morphologically.
3. The method of claim 2 wherein the analyte is an anti-inflammatory cytokine and the unlabeled primary antibody is an antibody that selectivity binds to an anti-inflammatory cytokine.