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H6: investigation summary: customer reported that product 337166 (bd multitest 6-color tbnk with trucount, ivd) lot 74827 is having tandem degradation in the vials.Customer provided fc data of apparent ¿bad¿ and ¿good¿ vials used.It was not known if ¿bad¿ vial was used successfully by customer on prior attempt or which lot is the apparent ¿good¿ vial.Additional information from customer was requested under child task pr# (b)(4) in support of investigation.Retain sample of subassembly 91-0717 batch 0064095 has been submitted for functional fc testing in plant 1149 in support of investigation and awaiting results as of (b)(6) 20.As per technical data sheet (tds) 23-10834-07 (rev.01), product 337166 (bd multitest 6-color tbnk with trucount, ivd) is an in vitro diagnostic (ivd) product.Intended use: bd multitest 6-color tbnk reagent with optional bd trucount tubes is a six-color direct immunofluorescence reagent for use with a suitably equipped bd flow cytometer to identify and determine the percentages and absolute counts of t, b, and natural killer (nk) cells, as well as the cd4 and cd8 subpopulations of t cells in peripheral blood.Summary and explanation: human lymphocytes can be divided into three major subset populations based on their biologic function and cell-surface antigen expression: t lymphocytes (cd3+), b lymphocytes (cd19+), and nk lymphocytes (cd16+cd56+).Bd multitest 6-color tbnk reagent can be used with or without bd trucount tubes to characterize these lymphocyte subset populations.When the reagent is used with bd trucount tubes, cytometer-specific bd software calculates lymphocyte subset percentages and absolute counts using flow data from the bd trucount beads and the sample.When the reagent is used without bd trucount tubes, cytometer-specific bd software calculates lymphocyte subset percentages directly from the sample flow data.If you enter lymphocyte data obtained from another instrument or method, the software can also calculate absolute counts.Precautions (tbnk reagent-specific): ¿ do not use reagent if you observe any change in appearance.Precipitation or discoloration indicates instability or deterioration.¿ the antibody reagent contains sodium azide as a preservative.However, take care to avoid microbial contamination, which can cause erroneous results.¿ do not use previously fixed and stored patient specimens.Whole blood samples refrigerated before staining can give aberrant results.Blast cells can interfere with test results.¿ use care to protect the tubes from direct light.Perform procedure at room temperature (20-25 °c).Storage and handling: ¿ store the reagent at 2-8 °c.Reagent in opened or unopended vials is stable until expiration date shown on the vial label.Do not use after this expiration date.¿ do not freeze the reagent or expose it to direct light during storage or incubation with cells.Keep the reagent vial dry.Evaluation of customer provided information: information captured from customer under parent pr# (b)(4) intake, include fc data of vials identified as ¿good¿ and ¿bad¿.Customer did not indicate if these ¿good¿ and ¿bad¿ vials are from different vials of same lot 74827 or different lots.Customer provided fc data show two sets of data, one named as ¿bad¿ and one ¿good¿ vial.It is observed the ¿bad¿ vial have excessive spillover on cd8 apc-cy7 vs cd19 apc plot from apc-cy7 into apc channel and cd3 fitc vs cd19 apc (double positive), not observed on the ¿good¿ vial.A similar spillover pattern, but to a lesser extent, is observed on the cd4 pe-cy7 vs cd16+56 pe plot from pe-cy7 to pe channel.This observed spillover suggests a possible breakdown of the tandem dyes possibly caused by excessive light or temperature exposure of reagent.Additionally, the observed double positive cd3+/cd19+ events could suggest having used previously stored blood specimens rather than freshly collected blood samples for staining and flow cytometry analysis, as described in the literature by eming et al.(cd3 positive b cells: a storage-dependent phenomenon, plos, 2014).Herein, the authors reported data findings in which t cell antigen cd3 can be substantially detected on surface of human b cells (which are cd20 and cd19 positive) after storage of blood specimens and this phenomenon depends on temperature and duration after blood collection from donors.This article describes that ex vivo cellular classification in peripheral blood might result in misleading interpretations when non-freshly collected blood samples are used for surface staining of t and b cell-specific markers, hence the importance to use freshly collected blood samples for staining and analysis of these lymphocyte surface antigens.It is known that product 337166 (bd multitest 6-color tbnk) reagent signal could be affected if used on previously fixed and later stored blood specimens (not freshly collected blood specimens), if exposed to direct light and/or stored out of recommended temperature (2-8 °c), hence such specific precautions, recommended handling and storage conditions are indicated in product tds 23-10834-07 (rev.01), precautions, and storage and handling sections.Request for additional information was initiated under child task (b)(4), in support of investigation.The following questions were asked to customer in these tasks: 1.Confirm if ¿bad¿ vs ¿good¿ fcs results are different vials of same lot 74827 or if ¿good¿ results was from a different lot.If different lot, indicate which lot # used resulting in ¿good¿ signal profile.2.Indicate how these reagent vials are handled (by end-users), including storage conditions, prior to and during use for routine staining.3.Was the ¿bad¿ vial previously used (open) by customer without observed issue of atypical staining showing apparent degradation? the following answers from customer were obtained: 1.The ¿good¿ and ¿bad¿ fcs files are from different vials of same lot 74827.2.The product is storage at 4ºc and protected from prolonged exposure to light.3.No, the vial had no visible degradation.Based on customer response, it is confirmed that ¿good¿ results were obtained using product 337166 lot 74827 in one (1) out of two (2) vials used by customer.The ¿bad¿ vial results correspond to a previously used (open) vial from customer of same product 337166 lot 74827, which suggest this used vial from customer could have been exposed to unfavorable conditions (i.E.Light or out of recommended storage temperature).Product 337166 (bd multitest 6-color tbnk) tds 23-10834-07 describes under precautions sections the specific cautions needed to avoid obtaining aberrant results.Retain sample fc test results: to confirm functional performance of 337166 (bd multitest 6-color tbnk with trucount, ivd) lot 74827, retain sample of subassembly 91-0717 batch 0064095 was tested thru flow cytometry (fc) analysis against reference 91-0717 batch 0240510.Fc data obtained show correct population profiles on cd3 fitc vs cd19 apc, cd8 apc-cy7 vs cd19 apc, cd8 apc-cy7 vs cd4 pe-cy7, and cd16+cd56 pe vs cd19 apc plots which were comparable to reference, and did not exhibit the aberrant populations or spillover observed by customer.In conclusion, retain results of subassembly 91-0717 batch 0064095 demonstrate correct function of product 337166 (bd multitest 6-color tbnk with trucount, ivd) lot 74827.Refer to attached tbnk retain functional testing for pr# (b)(4) pdf file.Product 337166 (bd multitest 6-color tbnk with trucount, ivd) lot 74827 manufactured from subassembly 91-0717 batch 0064095, was manufactured according to specifications.The review of the manufacturing device history record (bhr) or release records indicates product perform as intended per bd specifications and retain sample fc test results of subassembly 91-0717 batch 0064095 confirm product performs as intended.Based on investigation performed it is determined no further actions necessary and the claim is not confirmed.H3 other text : see h.10.
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