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Results from the Health and Retirement Study Biomarker Validation Project Eileen Crimmins Jung Ki Kim Heather McCreath Teresa Seeman DRAFT January 2013

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Table of Contents Aims 4 Approach of the Project 4 Analyses 5 Overview of Results 7 Comparison of Glycosylated Hemoglobin (HbA1c) from DBS and Whole Blood 11

General Information and Overview of Results 11 Descriptive Information 15 Figure 1: Scatter Plots of the Validation Samples 16 Figure 2: Bland Altman Plots 24 Figure 3: Differences between DBS and Venous Blood 30 Comparison of Whole Blood and DBS (including Regression Estimates and Z Converted Values)

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Figure 4: HbA1C in HRS Samples and NHANES and NSHAP 36

Comparison of Total Cholesterol from Dried blood Spots and Venous Blood 37 General Information and Overview of Results 37 Descriptive Information 38 Figure 1: Scatter Plots of the Validation Samples 41 Figure 2: Bland Altman Plots 46 Figure 3: Differences between DBS and Venous Blood 48 Comparison of DBS and Serum (including Regression Estimates and Z Converted Values)

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Figure 4: Total Cholesterol in HRS Samples and NHANES 53

Comparison of HDL Cholesterol from Dried blood Spots and Venous Blood 54 General Information and Overview of Results 54 Descriptive Information 55 Figure 1: Scatter Plots of the Validation Samples 58 Figure 2: Bland Altman Plots 63 Figure 3: Differences between DBS and Serum 65 Comparison of DBS and Serum (including Regression Estimates and Z Converted Values)

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Figure 4: HDL in HRS Samples and NHANES 70

Comparison of Cystatin C DBS and Venous Assays 71 General Information and Overview of Results 71 Descriptive Information 71 Figure 1: Scatter Plots of the Validation Samples 75 Figure 2: Bland Altman Plots 82 Figure 3: Differences between DBS and Serum 86 Comparison of DBS and Serum (including Regression Estimates and Z Converted Values)

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Figure 4: Cystatin C in HRS Samples and NHANES 94 Comparisons of CRP DBS and Venous Blood Assays 95

General Information and Overview of Results 95 Descriptive Information 96 Figure 1: Scatter Plots of the Validation Samples 102

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Figure 2: Bland Altman Plots 119 Figure 3: Differences in CRP by Serum Level 141 Comparison of DBS and Serum (including Regression Estimates and Z Converted Values)

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Figure 4: CRP in HRS and NHANES 167

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The aims of this project were to

a. Calibrate DBS results against whole blood values. b. Compare DBS across labs c. Compare across filter papers.

The Approach of the Project: To accomplish our aims, we

a. Collected dried blood spots and venous blood from 92 non-sample subjects aged 50 + during the period from June through December 2010. These were volunteers who came to the UCLA Alhambra Research Center. Participants provided signed consent to be part of the study and were paid $25 to participate.

b. DBS were sent to a variety of labs: Heritage, the University of Vermont, the University of Washington, Geonostics (using FlexSite assay), and to Thom McDade.

c. HbA1c was assayed from fresh whole blood at a local lab. d. For other assays, whole blood was separated into serum and plasma.

Serum was frozen and sent to the University of Vermont for assay of total and HDL cholesterol, CRP, and Cystatin C.

The DBS were sent to the following Laboratories:

a. Heritage DBS Cards. These cards were sent to HRS in the mail (not frozen) and then sent from HRS to Heritage with HRS survey subject cards for assay of HbA1c, Total cholesterol, and HDL cholesterol. These samples were exposed to conditions similar to those of the actual HRS samples.

b. DBS cards for CRP and Cystatin C were sent directly to the University of Vermont for assay (these were frozen at the UCLA facility and sent frozen to Vermont in one batch).

c. We also collected one blood spot on a Biosafe treated card that we sent to Geonostics (which has acquired FlexSite). These assays were done in the Spring of 2012. The cards were sent frozen from UCLA where they were stored at -70 degrees from the time of collection until just before assay.

d. We also sent DBS spots on untreated cards (Biosafe card) to the University of Washington for examination of Total cholesterol, HDL cholesterol, HbA1c, CRP and Cystatin C. These cards were frozen after drying and they were sent frozen to Washington. Most of the assays were done in 2011. However, washington developed and validated the CRP and Cystatin C assays during the course of this project. The CRP and Cystatin C assays were done in August of 2012.

e. We sent one frozen untreated Biosafe card to Thom McDade for assay of CRP for comparison with assays done by the University of Vermont.

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Comparisons that can be made a. Comparisons can be made between whole blood values of HbA1c and

DBS assays from the University of Washington, Heritage, and Geonostics (using FlexSite assay).

b. Serum blood comparisons can be made with DBS assays for Total and HDL cholesterol from the University of Washington and Heritage labs.

c. DBS assays for the University of Washington and Heritage can be compared for HbA1c, total, and HDL cholesterol.

d. Comparisons of CRP assays from DBS and serum values can be done for the University of Vermont, Thom McDade’s lab and the University of Washington. These three DBS assays can also be compared to each other.

e. Cystatin C from serum at the University of Vermont is compared to the assays from DBS at the University of Vermont, and the University of Washington.

f. Values for DBS assays from two cards - the Heritage Card and what we call the Biosafe card (cards from earlier HRS rounds) - can be compared for CRP and Cystatin C assayed at the University of Vermont.

Analyses Presented in this Document: We present detailed information for each of the assays in a separate section: HbA1c, Total cholesterol, HDL cholesterol, CRP, and Cystatin C. For each of the assays we present some general information, descriptive statistics and information, followed by a variety of methods to assess comparability between assays. The specific information included in each section is arranged as follows:

1. Descriptive information on the assays for all the available data followed by descriptive information on matched pairs of assays from DBS and venous assays

a. The percent who would be scored in the level of risk using conventional definitions of risk

b. The distribution of values on the validation samples

c. Scatterplots relating the DBS and venous assays and DBS assays.

d. Bland Altman Plots which provide conventional ways of comparing values from multiple assays. This comparison and the one below help to determine whether the difference between assays is similar across the range of values.

Bland Altman plots the Difference between two assays on the Y axis against the Average on the X Axis. For our assays the formulas are

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Average = (DBS + Whole Blood)/2; Difference = DBS - Whole blood. We show the 95% confidence intervals for the difference.

e. Differences between DBS and Venous Blood by level of venous blood assay

2. We then investigate methods of transforming DBS values.

a. Quartile distributions of venous and DBS assays to determine how similarly values from different assays would distribute by quartile.

b. Transforming DBS scores into conventional whole blood scores 1) Comparisons of assays when DBS are converted to whole blood values using Z scores. We can use Z scores to transform the values of one assay into those based on some standard and retain the variability. If we compute the Z score for one assay, (Z 1= x mean1/ sd1), we can estimate the value of x in a standard assay by substituting the Z1 value into an equation using the mean and standard deviation of the distribution from the standard assay (Z1 sds) + means= xs

The xs value reflects the value that would be obtained with the standard assay.

2) Regression equations relating the whole blood and venous blood assays and the multiple DBS assays when available. One could estimate a venous equivalent value using a regression equation based on comparing values from a standard assay and another assay. This may be a good solution if the variance explained is very high. If the variance explained is not high, it results in losing variability as unexplained variance results in estimates closer to the mean.

3) Comparison of assays when the regression equations shown in section 9 are used to estimate DBS values.

3. Comparison of HRS sample assays with assay results from other surveys

At the end of each section, we compare HRS sample assay results with those from other surveys with similar assays.

4. Results: We include conclusions throughout these analyses but we summarize the high points below.

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Overview of Results 1. HbA1c

All the DBS values are reasonably highly correlated with the venous blood values (R2 of .74 for FlexSite, .95 for the University of Washington, and .96 for Flexite). The University of Washington generally seems to be a superior assay with the exception that in the Bland Altman results which indicate that the differences between the UWDBS assay for HbA1c and the venous blood assay vary with the level of the assay. The R2 between the two DBS assays is .71. The Flexite assay also seems to match venous values very well: Flexite is highly correlated with the venous (R2 of 0.93). The R2 between Flexite and UWDBS is very high (R2 of 0.95). The regression estimated equivalent of venous values and the Z score estimated values from the University of Washington and FlexSite have average values very close to whole blood. Because the R2 is so high the regression transformation is reasonable although we would be cautious in using transformed values. Using cutoffs is an issue even when the R2 is high. Transforming the Heritage assay values using the regression equation results in a narrowing of the range. We need to note that the Heritage samples were not maintained under the same conditions as the University of Washington samples. The Heritage samples were shipped in the mail like regular HRS samples which could be the reason that the assay does not relate as strongly to serum levels. Examination of the HRS data show that the various assays appear to be relatively similar in distribution. However, where they differ is right at the cutpoint for high risk. This may need to be a special focus of any adjustment. The University of Washington provided whole blood equivalent values differs more from the whole blood values we obtained than any of the DBS assays. It indicates levels of dysregulation that are too high. 2. Total Cholesterol.

The assays of total cholesterol produce very different values. The University of Washington value is very high relative to the value from the venous blood and the Heritage DBS. The Heritage value is closer to the venous value, although somewhat lower than venous on average. The University of Washington distribution of DBS assays shows that there were a significant number of very high values in their assay. Using traditional cutpoints will not work with the University of Washington values. While the mean level of the University of Washington differs more from the venous level than the Heritage mean difference, the University of Washington DBS is more closely associated with venous blood values than the Heritage DBS

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values are (R2 .55 versus .30). The relationship between the University of Washington and the Heritage values is very low (R2 .23). The size of the differences between both DBS assays and the value of the venous assay differs by the level of the assays. Using a regression approach to produce serum cholesterol equivalents does not work well because of the low associations. Z scores are more promising for creating equivalent values. The DBS cholesterol assays need to be improved or replaced. The lack of strong association between the cholesterol values for the two HRS labs is going to make it challenging to examine change over time. Examination of HRS data indicates that the distribution from Biosafe assays for 2006 and 2006 are quite different. The 2006 Biosafe assay is similar to NHANES and the 2008 assay is similar to the University of Washington in distribution, with the exception of some very high values in the Washington assay. 3. HDL cholesterol

The HDL values are similar to the Total cholesterol values in that the University of Washington assay produces values much higher than the others. On the other hand, the association between the University of Washington DBS values and the venous blood values is stronger (.48) than that of Heritage DBS (.32). The association of the two DBS assay values is low (.20). Z scores may be a better method of transformation than regression estimates of venous equivalents for these measures. The low value of association between the two HRS labs is going to make examining change over time difficult. The distribution of the HRS 2006 values and 2008 values from Biosafe are similar to each other and to NHANES. The University of Washington value is shifted to the right. The Biosafe distribution in 2006 for HRS is lower than that for Biosafe in 2008. 4. Cystatin C

The mean of the serum values is considerably higher than that of the DBS values but the correlation of the serum and DBS values is fairly high (~.76 - .80). The correlation between the DBS assays based on the two cards both done at the Univ of Vermont is even higher (.90) and the means are almost identical. The somewhat smaller value for the Heritage DBS card could be related to either the handling of the sample or to the card itself. The correlation of the DBS asays at the Univ of VT and Washington are relatively high. Assays using the same card and same storage are correlated .91 to .96. The University of Washington lab provided two assays for each sample. Mean values of the two Washington DBS are much closer to that of the serum values, particularly the second Washington DBS.

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The serum value from the Univ of Vermont assay does not have the same range as Cystatin C assays in the literature. It has much lower values and conventional cutoffs for high risk cannot be used. The moderately strong association between the DBS and serum values means that we could use either the regression or the Z score approach to get a value very close to the value produced by the Serum Cystatin C assay; however, because this assay value is quite different from those reported in the literature, the value of doing this is not clear. The distribution of Cystatin C values in HRS is almost the same in 2006 and 2008 but it is very different from that in NHANES.

5. C-Reactive Protein

The values of the Vermont serum assays differ from the DBS assays of CRP done at Vermont (called the Heritage and Biosafe for the cards used), the McDade assay, and the Washington values. The Vermont Serum assay has a relatively low value, the VT DBS values are even lower, the Washington values are highest. . The associations between the DBS assays done at Vermont and the venous blood assays are generally very high (correlation coefficients .98-.99). The UW2 with a higher range is also realtively highly correlated with the serum values (.86). The differences between the DBS and serum values are larger at higher levels. When both values are log transformed, as is often the case in CRP analysis, the differences are similar across the assay range.

The McDade DBS assay and the Vermont DBS assay have very different values and ranges. McDade’s assay detects much lower values but it has a severely restricted range at the top. However, the correlation between the two sets of DBS assays is high. When cases are imputed for the Vermont assay, the correlation is .89. The Washington 1 assay tends to have lower association with other values. It has 3 values that are coded as outside of the range (too high and are not assigned an actual assay value). For assay 2, these three samples were diluted 1:2 or 1:4 and then re-assayed. The second Washington assay has a value for those three cases, which are very high, all over 20. The Washington values are differentially correlated with the venous blood assay values because of this; correlation with Washington 2 is high (.87) but that with Washington 1 is considerably lower (.56).

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Because the relationships among many of these assays are reasonably high, various approaches to converting them into similar metrics should work reasonably well. The distribution of the CRP values in HRS in 2006 and 2008 is very similar. The values are lower than those in NHANES and similar to those in NSHAP. Sum of Overall Results: The DBS approach appears to do quite well for indicating levels of HbA1c, Cystatin C, and C-Reactive Protein. Assessing lipid levels continues to be a challenge.

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Comparison of Glycosylated Hemoglobin (HbA1c) from DBS and Whole Blood General Information HbA1c was measured using DBS at Heritage, the University of Washington, and. Geonostics (based on FlexSite assay). Whole blood assays were done for 66 subjects at a local AMA commercial lab. Heritage assays were collected using the Heritage card. Heritage was sent 92 cards (Bloodspots collected as numbers 1-6). Values were returned values for 89 cases (3 ID problems). University of Washington assays were collected using the untreated Biosafe card. UW was sent 91 cards. These samples were from drops 7–12. No data were returned for 14 cards. There was no sample for 5 and either a very small or a smeared spot for 9 cards. So 9/86 had an unusable sample and there were 77 usable samples. Of these, some were classified as very small, smeared, or of poor quality. Washington suggests doing the comparison without the very small (25), smeared (19), poor quality (1). When we ran the mean for the two groups (37 good and 39 bad), there is no significant difference (when we eliminate 1 outlier with a value of 10). UW produces a whole blood equivalent value which we find not to be very useful but we include it in the tables. FlexSite was sent 90 cards which had been frozen for some time while the company was still recovering from the bankruptcy arrangements. These cards did not have pretreated spots. Overview of Results All of the DBS values are reasonably highly correlated with the venous blood values (R2 of .74 for Heritage, .95 for the University of Washington and .96 for FlexSite). The means of the three DBS assays are fairly similar but the range of the University of Washington assay is less. The University of Washington and Flexite appear to be marginally superior assays with the exception of the Bland Altman results which indicate that the differences between the UWDBS assay for HbA1c and the venous blood assay vary with the level of the assay. The regression estimated equivalent to venous values and the Z score estimated values from the University of Washington and FlexSite result in

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means very close to whole blood. Because the R2 is so high the transformation is reasonable although we would be cautious in doing this. Using cutoffs is an issue even when the R2 is high. Transforming the Heritage assay results in a narrowing of the range. We need to note that the Heritage samples were not kept under the same conditions as the University of Washington samples.

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1. Descriptive Information on All HbA1c Assays

HbA1c Average Range #Cases Interquartile

Range UCLA Whole Venous Blood 5.89 4.5-10.7 66 0.6 Heritage DBS 6.13 5.2-13.8 89 0.7 UWDBS 6.08 5.0-10.0 77 0.4 UWEQ 6.13 4.8-11.1 77 0.5 FlexSite 6.05 4.7-12.4 90 0.6

Mean values – The DBS means from Heritage, Washington and Flexite are significantly (DBS-venous) higher than the venous (Ns Heritage = 64, UW=58, Flexite=66). UW Whole blood equiv difference from venous is even larger. (N=58) UW Whole blood equiv and Heritage DBS are not significantly different (N=75).

2. %High HbA1c (>=6.4) with no adjustment to data % N UCLA whole blood (N=66) 15.15 10 Heritage (N=89) 25.84 23 U Washington (N=77) 16.88 13 U Washington Equivalent (N=77) 23.38 18 FlexSite (N=90) 15.56 14 High Values – The percentage high in the Univ of Washington and the FlexSite assays is similar to that from whole blood. The Heritage assays produce a level much higher; similar to that in the Univ of Washington Whole blood equivalent.

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3. Distribution of HBA1C (%) in the Validation Samples

Washington Washington Equivalent

UCLA whole blood

Heritage FlexSite

<5.0 0.00 1.30 3.03 0.00 1.11 5.0-5.2 1.30 0.00 3.03 0.00 1.11 5.2-5.4 0.00 2.60 4.55 4.00 6.67 5.4-5.6 2.60 6.49 22.73 10.67 18.89 5.6-5.8 18.18 22.08 16.67 0.00 15.56 5.8-6.0 24.68 14.29 18.18 25.33 20.00 6.0-6.2 19.48 19.48 7.58 17.33 12.22 6.2-6.4 16.88 10.39 9.09 12.00 8.89 6.4-6.6 7.79 10.39 6.06 9.33 4.44 6.6-6.8 1.30 3.90 0.00 8.00 2.22 6.8-7.0 3.90 1.30 1.52 8.00 0.00 7.0+ 3.90 7.79 7.58 5.33 8.89 There are no very low values in the DBS assays done by the University of Washington and Heritage. The range of is these DBS assays is smaller than that of the whole blood assays. The FlexSite range is more similar to the whole blood range. FlexSite has the most very high assays.

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4. Descriptive Information on matched DBS and Venous blood samples for HbA1c HBA1C Average S.D. Range #Cases Correlation Coeff. UCLA whole blood 5.84 0.51 4.7-7.3 64 0.85950

<.0001 Heritage 5.99 0.49 5.2-7.6 64 <.0001 UCLA whole blood 5.92 0.84 4.5-10.7 58 0.97313

<.0001 UWDBS 6.09 0.64 5.0-10.0 58 <.0001 UCLA whole blood 5.92 0.84 4.5-10.7 58 0.97313

<.0001 UWEQ 6.15 0.80 4.77-11.06 58 <.0001 UCLA whole blood 5.89 0.80 4.5-10.7 66 0.96435 FlexSite 5.96 0.82 4.7-11.2 66 <.0001 <.0001 Heritage 6.07 0.50 5.2-7.6 75 0.84448 UWDBS 6.04 0.35 5.4-7.0 75 <.0001 .4570 Heritage 6.13 0.96 5.2-13.8 87 0.93455 FlexSite 5.96 0.85 5.0-12.4 87 <.0001 <.0001 Flexite 5.95 0.80 4.7-11.2 76 0.97478 UWDBS 6.08 0.59 5.0-10.0 76 <.0001 <.0001 The results for the matched samples are similar to those reported above. The DBS values are higher than the whole blood values and the UWEQ is even higher than the DBS. The two DBS mean vales are statistically the same although the range of the University of Washington value is smaller.

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Figure 1. Scatter Plots of the Validation Samples: Whole Blood (UCLA lab) versus DBS Heritage

UCLA lab: Hemoglobin A1c

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Heritage lab: Hemoglobin A1c

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Heritage against UCLA lab

Number of Observations Read 95 Number of Observations Used 64 Number of Observations with Missing Values 31 R-Square 0.7387 Whole Blood (hrsa1c) = 0.524121 + 0.887383* DBS Heritage (hdba1c)

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UCLA lab: Hemoglobin A1c

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UW: A1c (%)

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Washington against UCLA lab

Number of Observations Read 95 Number of Observations Used 58 Number of Observations with Missing Values 37 R-Square 0.9470 Whole Blood (hrsa1c) = -1.904125 + 1.282996*DBS Univ of Washington (uwa1c)

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UCLA lab: Hemoglobin A1c

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UW: A1c whole blood equivalent (%)

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Washington equivalent against UCLA lab

Number of Observations Read 95 Number of Observations Used 58 Number of Observations with Missing Values 37 R-Square 0.9470 Whole Blood (hrsa1c) = -0.352793 + 1.019586* Univ of Wash Equivalent (uwa1ceq)

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UCLA lab: Hemoglobin A1c

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HBA1C_FLEXITE

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Flexite against UCLA lab

Number of Observations Read 95 Number of Observations Used 66 Number of Observations with Missing Values 29 R-Square 0.9300 Whole Blood (hrsa1c) = 0.28797 + 0.94046 * Flexite (hba1c_flexite)

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Heritage lab: Hemoglobin A1c

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UW: A1c whole blood equivalent (%)

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Washington Equivalent against Heritage

Number of Observations Read 95 Number of Observations Used 75 Number of Observations with Missing Values 20 R-Square 0.7131 Heritage DBS (hdba1c) = 0.287954 + 0.950092* Univ of Wash Equiv (uwa1ceq)

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Comparison of Two DBS Values

Heritage lab: Hemoglobin A1c

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UW: A1c (%)

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Number of Observations Read 95 Number of Observations Used 75 Number of Observations with Missing Values 20 R-Square 0.7131 Heritage DBS (hdba1c) = -1.157641 + 1.19555* Univ Wash DBS (uwa1c)

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Heritage lab: Hemoglobin A1c

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Flexite against Heritage

Number of Observations Read 95 Number of Observations Used 87 Number of Observations with Missing Values 8 R-Square 0.8734 Heritage DBS (hdba1c) = -0.19810 + 1.06181 * FlexSite (hba1c_flexite)

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UW: A1c (%)

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Flexite against Washington

Number of Observations Read 95 Number of Observations Used 76 Number of Observations with Missing Values 19 R-Square 0.9502 Washington DBS (uwa1c) = 1.80515 + 0.71781 * FlexSite (hba1c_flexSite)

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Figure 2. HbA1c Bland Altman Plots Difference between Heritage and Whole Blood Average= (Heritage + Whole Blood)/2 Difference = Heritage - Whole blood (hdba1c - hrsa1c);

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Mean 95% CI Average 5.91 5.79-6.03 Heritage – Whole Blood 0.15 0.08-0.22 # of out of range (2SD): 2 The range is wide; few outside the range; the difference appears similar across the range.

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Difference between U Washington DBS and Whole Blood Average= (UWDBS + Whole Blood)/2 Difference = UWDBS - Whole blood (uwa1c-hrsa1c);

Difference

-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0

Average4 5 6 7 8 9 10 11

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Mean 95% CI Average 6.01 5.82-6.20 UWash – Whole Blood 0.18 0.11-0.25 # of out of range (2SD): 3 The range is larger than for Heritage; the number outside the range is similar; however, the difference changes with the level. At low values the difference is positive; at high values it is negative.

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Difference between FlexSite and Whole Blood Average= (FlexSite + Whole Blood)/2 Difference = FlexSite - Whole blood (hba1c_flexSite - hrsa1c);

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Mean 95% CI Average 5.92 5.73-6.12 FlexSite – Whole Blood 0.07 0.01-0.12 # of out of range (2SD): 4

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Difference between Heritage DBS and U Washington DBS Average= (Heritage + UWDBS)/2 Difference = Heritage - UWDBS (hdba1c – uwa1c);

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Mean 95% CI Average 6.05 5.96-6.14 Heritage – UWash 0.024 -0.04 ~ 0.08 # of out of range (2SD): 2 The University of Washington value is greater at lower average levels.

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Difference between Heritage and FlexSite Average= (Heritage + FlexSite)/2 Difference = Heritage - FlexSite (hdba1c - hba1c_flexSite);

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Mean 95% CI Average 6.04 5.86-6.23 Heritage – Flexite 0.17 0.10-0.24 # of out of range (2SD): 3

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Difference between U Washington DBS and FlexSite Average= (UWDBS + FlexSite)/2 Difference = UWDBS - FlexSite (uwa1c - hba1c_flexSite);

Difference

-2.0-1.8-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0

Average4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0

HBA1C

Mean 95% CI Average 6.02 5.86-6.17 UWDBS – FlexSite 0.13 0.07-0.18 # of out of range (2SD): 4 At low values the difference is positive; at high values it is negative.

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Figure 3. Differences between DBS and Venous Blood by level of venous blood

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We compare a number of ways to use the DBS values – as alternatives to the assayed value – and compare these to values from whole blood. Quartile of HbA1c: Heritage - Whole blood (N=64) Heritage whole 1st qt 2nd qt 3rd qt 4th qt 1st qt 23.44

(N=15) 6.25

(N=4) 3.13

(N=2) 0.0

(N=0) 2nd qt 9.38

(N=6) 4.69

(N=3) 3.13

(N=2) 0.0

(N=0) 3rd qt 3.13

(N=2) 12.50 (N=8)

7.81 (N=5)

3.13 (N=2)

4th qt 0.0 (N=0)

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3.13 (N=2)

20.31 (N=13)

Cutpoints for Quartiles 1st 2nd 3rd Heritage ~5.7 ~5.9 ~6.2 Whole Blood ~5.5 ~5.75 ~6.1 About 56% are in the same quartile of the Heritage and whole blood values. High and low are closest. Quartile of HbA1c: Univ Washington DBS-Whole blood (N=58) Wash whole 1st qt 2nd qt 3rd qt 4th qt 1st qt 27.59

(N=16) 5.17

(N=3) 0.0

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(N=0) 2nd qt 5.17

(N=3) 18.97

(N=11) 6.90

(N=4) 0.0

(N=0) 3rd qt 0.0

(N=0) 5.17

(N=3) 6.90

(N=4) 3.45

(N=2) 4th qt 0.0

(N=0) 0.0

(N=0) 3.45

(N=2) 17.24

(N=10) Cutpoints for Quartiles 1st 2nd 3rd U Washington ~5.8 ~6.0 ~6.2 Whole Blood ~5.5 ~5.8 ~6.2 About 70% of the DBD and whole blood assays are in the same quartile. The University of Washington does better than Heritage in matching quartiles

32

Quartile of HbA1c: FlexSite - Whole blood (N=66) Flexite whole 1st qt 2nd qt 3rd qt 4th qt 1st qt 24.24

(N=16) 3.03

(N=2) 0.0

(N=0) 0.0

(N=0) 2nd qt 7.58

(N=5) 10.61 (N=7)

6.06 (N=4)

1.52 (N=1)

3rd qt 1.52 (N=1)

3.03 (N=2)

15.15 (N=10)

3.03 (N=2)

4th qt 0.0 (N=0)

0.0 (N=0)

4.55 (N=3)

19.70 (N=13)

Cutpoints for Quartiles 1st 2nd 3rd FlexSite ~5.5 ~5.8 ~6.1 Whole Blood ~5.5 ~5.8 ~6.2 About 70% of the whole blood assays are in the same quartile, similar to the University of Washington.

33

Equations relating whole blood HbA1c with DBS and Three DBS values to each other:

HbA1c EQ y a b x R2 N Whole blood -1.904 +1.2830 UWDBS 0.95 58 Whole blood 0.5241 +0.8874 Heritage DBS 0.74 64 Whole blood -0.352783 -1.018586 UWEQ 0.95 58 Whole blood 0.28797 +0.94046 FlexSite 0.93 66 Heritage DBS -1.1576 +1.1956 UW DBS 0.71 75 Heritage DBS 0.287854 +0.850082 UWEQ 0.71 75 Heritage DBS -0.19810 +1.06181 FlexSite 0.87 87 FlexSite -2.09303 +1.32375 UWDBS 0.95 76 UWDBS 1.80515 +0.71781 Flexite 0.95 76

The University of Washington and FlexSite DBS values match venous blood values better than Heritage values; however the FlexSite and UWDBS (R2=.95) and the Heritage and FlexSite DBS values are more highly correlated (R2 = .87) than the Heritage and UWDBS (R2=.71). Conversions of DBS based on regression equations It is possible to use the regression equation to estimate whole blood values based on the DBS value. When this is done the estimated means, SD, and range are closer to those of the whole blood. The percent with high levels is somewhat lower. Mean SD Range %High Risk

(>=6.4) Whole Blood 5.92 0.84 4.5-10.7 15.5 Heritage Est of Serum Equiv 5.84 0.44 5.1-7.3 10.9 Mean SD Range %High Risk

(>=6.4) Whole Blood 5.92 0.84 4.5-10.7 15.5 UW Est of Serum Equiv 5.92 0.82 4.5-10.9 13.8 Mean SD Range %High Risk

(>=6.4)

34

Whole Blood 5.92 0.84 4.5-10.7 15.5 FlexSite Est of Serum Equiv 5.89 0.77 4.7-10.8 13.6 Estimating values with the regression equation results in relatively close means for DBS values. The range of values and the S.D. of values for the U of Washington and FlexSite look similar to those in the whole blood. The percentage high is slightly lower. Using the regression on the Heritage values, reduces their range to only 2.2 points; the SD is about half of that in the other distributions; and results in a relatively low estimate of the percent high.

35

An alternative approach is to convert DBS values to whole blood equivalent values based on Z scores. We compute z scores in the two distributions and match equivalent Z scores and then transform DBS values into whole blood values.

HBA1C Average S.D. Range #Cases %high risk (>=6.4)

UCLA whole blood 5.84 0.51 4.7-7.3 64 14.1%(N=9) Heritage Converted to UCLA 5.84 0.51 5.0-7.5 64

12.5%(N=8)

UCLA whole blood 5.92 0.84 4.5-10.7 58 15.5%(N=9) Biosafe(Washington) Converted to UCLA 5.92 0.84 4.5-11.1 58

13.8%(N=8)

UCLA whole blood FlexSite Converted to UCLA

5.89 5.89

0.80 0.80

4.5-10.7 4.7-11.0

66 66

15.2%(N=10) 13.6%(N=9)

The percentage with high levels of glycosylated hemoglobin (>=6.4%)

(N=64) Whole blood 14.1% Heritage DBS assay original 21.9% Heritage DBS used in regression est 10.9% Heritage DBS assay converted using Z scores 12.5% (N=58) Whole blood 15.5% UW DBS assay original 17.2% UW DBS used in regression est 13.8% UW DBS assay converted using Z scores 13.8% (N=66) Whole blood 15.2% FlexSite assay original 13.6% FlexiSte used in regression est 13.6% FlexSite assay converted using Z scores 13.6%

36

Figure 4. HbA1C in HRS Samples and NHANES and NSHAP HRS Sample (2006, 2008 two labs), NHANES, NSHAP (Weighted)

The distribution of values is generally similar for HRS 2006 done by Biosafe and HRS 2008 by Biosafe and both of these are quite similar to NHANES venous values . There is a shift in the modal value in the FlexSite assays which is similar to the value in NSHAP.

37

Comparison of Total Cholesterol from Dried Blood Spots and Venous Blood

1. Dried blood spots were assayed at

1) Heritage labs from DBS on heritage cards (N=87). The mean was 177 (range is 114-279).

2) University of Washington assayed DBS based on Biosafe cards (N= 91). Spot quality essentially the same as for HbA1c: 1 spot is said to be out range; 50 not good quality (mean is 281); 41 with good quality (mean is 261).

3) University of Washington provides a Plasma equivalent (mean 161 – range 95 – 244).

Venous blood –

Serum assayed at the University of Vermont lab (N=94) - range is 110 to 285 mean is 191

Overview of Results Differences between assays in level are significant. The University of Washington value is very high relative to venous and Heritage. Heritage value is closer to venous but lower than venous. The University of Washington distribution of DBS assays shows that there were a significant number of very high values. Using traditional cutpoints will not work with the University of Washington values. While the mean level of the University of Washington differs more from the venous level than the Heritage mean difference, the University of Washington DBS is more closely related to venous blood values than Heritage DBS (R2 .55 versus .30). The relationship between the University of Washington and the Heritage values is very low (R2 .23). Differences between both DBS assays and the venous assays differ by the level of the assays. Using a regression approach with Cholesterol to produce Serum equivalents does not work because of the low associations. Z scores are more promising.

38

1. Descriptive Information on All Total Cholesterol Assays Cholesterol- total Average Range #Cases Interquartile

VT Serum 191.13 110-285 94 48 DBS Heritage 177.33 114-279 87 39 DBS UW 272.42 167-403 91 73 DBS UWPlaEQ 161.35 95-244 91 46

2. %High Total Cholesterol (>=240) – no adjustments % N Vermont serum (N=94) 8.51 8 Heritage (N=87) 4.6 4 Washington (N=91) 74.73 68 Washington Equivalent (N=91)

1.1 1

The mean values for UWDBS are almost 100 points higher than the values for Heritage DBS. The Univ of Washington Plasma Equivalent value is the lowest of all. Using conventional cutoff levels 2/3 of the sample is high according to the Univ of Washington DBS assay and almost no one is using the Heritage assay or the plasma equivalent provided by the UW.

39

3. Distribution of Total Cholesterol (%)

Washington DBS

Washington Equivalent

Heritage DBS Vermont Serum

-120 15.38 1.15 2.13 120-130 5.49 2.30 4.26 130-140 3.30 4.60 2.13 140-150 9.89 10.34 5.32 150-160 10.99 11.49 6.38 160-170 1.10 16.48 16.09 8.51 170-180 3.30 9.89 13.79 6.38 180-190 2.20 8.79 12.64 13.83 190-200 6.59 6.59 6.90 12.77 200-210 2.20 6.59 5.75 9.57 210-220 5.49 4.40 3.45 9.57 220-230 2.20 1.10 3.45 6.38 230-240 2.20 0.00 4.60 4.26 240-250 6.59 1.10 2.30 2.13 250-260 5.49 0.00 0.00 3.19 260-270 7.69 0.00 0.00 1.06 270-280 8.79 0.00 0.00 0.00 280-290 7.69 0.00 0.00 2.13 290-300 7.69 0.00 0.00 0.00 300+ 30.77 0.00 0.00 0.00 The lowest values in the Univ of Washington DBS distribution are in the 160s – which is close to the midpoint of the Heritage or the University of Vermont distribution.

40

4. Descriptives on matching samples for Total Cholesterol Descriptives

Cholesterol Average S.D. Range #Cases Correlation

Coeff. VT Serum 191.38 35.52 110-285 86 0.55048

<.0001 DBS Heritage 177.14 30.79 114-279 86 <.0001 VT Serum 190.22 35.34 110-285 90 0.74117

<.0001 DBS UW 273.02 52.68 166.94-402.76 90 <.0001 VT Serum 190.22 35.34 110-285 90 0.74117

<.0001 DBS UWPlaEQ 161.73 33.30 94.68-243.73 90 <.0001

The differences are the same as those described above.

41

Figure 1. Scatter Plots of the Validation Samples for Total cholesterol from Serum and DBS at Heritage

Vermont: Cholesterol (mg/dL)-Serum

0

50

100

150

200

250

300

Heritage lab: Cholesterol

0 50 100 150 200 250 300

Heritage against Vermont

Number of Observations Read 95 Number of Observations Used 86 Number of Observations with Missing Values 9 R-Square 0.3030 Serum (uvchol) = 78.88284 + 0.635098* Heritage DBS (hdbchol)

42

Vermont: Cholesterol (mg/dL)-Serum

0

50

100

150

200

250

300

350

400

450

UW: Cholesterol (mg/dL)

0 50 100 150 200 250 300 350 400 450

Washington against Vermont

Number of Observations Read 95 Number of Observations Used 90 Number of Observations with Missing Values 5 R-Square 0.5493 Serum (uvchol) = 54.48611 + 0.497157* Univ of Washington DBS (uwchol)

43

Comparison of two DBS

Heritage lab: Cholesterol

0

50

100

150

200

250

300

350

400

450

UW: Chol plasma equivalent conc (mg/dL)

0 50 100 150 200 250 300 350 400 450

Washington Equivalent against Heritage

Number of Observations Read 95 Number of Observations Used 86 Number of Observations with Missing Values 9 R-Square 0.2272 Heritage DBS (hdbchol) = 106.5818 + 0.438086* Univ of Wash EQ (uwcholeq)

44

Vermont: Cholesterol (mg/dL)-Serum

0

50

100

150

200

250

300

350

400

450

UW: Chol plasma equivalent conc (mg/dL)

0 50 100 150 200 250 300 350 400 450

Washington Equivalent against Vermont

Number of Observations Read 95 Number of Observations Used 90 Number of Observations with Missing Values 5 R-Square 0.5493 Serum (uvchol) = 63.00789 + 0.786572* Univ of Washington EQ (uwcholeq)

45

Heritage lab: Cholesterol

0

50

100

150

200

250

300

350

400

450

UW: Cholesterol (mg/dL)

0 50 100 150 200 250 300 350 400 450

Washington against Heritage

Number of Observations Read 95 Number of Observations Used 86 Number of Observations with Missing Values 9 R-Square 0.2272 Heritage DBS (hdbchol) = 101.8355 + 0.276895* Univ of Washington DBS(uwchol)

46

Figure 2. Total Cholesterol Bland Altman Plots Difference between Heritage DBS cholesterol and U Vermont Serum Average = (HeritageDBS + UVcholserum)/2 Difference = (HeritageDBS – uvcholserum)

Difference

-120-100-80-60-40-20

0204060

Average100 120 140 160 180 200 220 240 260 280 300

Total Cholesterol

Mean 95% CI Average 184.26 179.08-189.44 Heritage – Vermont Serum -14.24 -19.87~ -8.61 # of out of range (2SD): 4 The differences are larger at higher values meaning that at higher values the assay is less precise. At higher values, the Heritage assay underestimates are greater.

47

Difference between UWashington DBS cholesterol and U Vermont Serum Average = (UWcholDBS + UVsholserum)/2 Difference = uwcholDBS – uvcholserum

Difference

-200

20406080

100120140160

Average100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420

Total Cholesterol

Mean 95% CI Average 231.62 223.11 - 240.13 UWash – Vermont Serum 82.80 75.45 – 90.15 # of out of range (2SD): 5 If the value of the assay is low, the difference is smaller; if the value if large, the difference is larger.

48

Figure 3. Differences between DBS and Venous Blood by Level of Cholesterol in Vermont assay

-150

-100

-50

0

50

100

150

200

0 50 100 150 200 250 300

UV-HeritageUV-Washington UV-WashingtonEq

Differences are all positive for Univ of Washington and mostly negative for Heritage. The larger differences at higher levels are shown here also.

49

Quartile Values for Heritage DBS and UVT Serum: Total Cholesterol (N=86) Heritage uv 1st qt 2nd qt 3rd qt 4th qt 1st qt 13.95

(N=12) 5.81

(N=5) 5.81

(N=5) 1.16

(N=1) 2nd qt 6.98

(N=6) 6.98

(N=6) 8.14

(N=7) 2.33

(N=2) 3rd qt 1.16

(N=1) 6.98

(N=6) 5.81

(N=5) 10.47 (N=9)

4th qt 3.49 (N=3)

4.65 (N=4)

5.81 (N=5)

10.47 (N=9)

Cutpoints for Quartiles 1st 2nd 3rd Heritage ~155 ~171.5 ~193 UV ~168 ~191 ~215 Just over a third (37%) are in the same quartile. Quartile Values for UWcholesterolDBS and UVTSerum (N=90) Uw uv 1st qt 2nd qt 3rd qt 4th qt 1st qt 16.67

(N=15) 6.67

(N=6) 2.22

(N=2) 1.11

(N=1) 2nd qt 5.56

(N=5) 7.78

(N=7) 6.67

(N=6) 3.33

(N=3) 3rd qt 3.33

(N=3) 7.78

(N=7) 8.89

(N=8) 6.67

(N=6) 4th qt 0.0

(N=0) 2.22

(N=2) 7.78

(N=7) 13.33

(N=12) Cutpoints for Quartiles 1st 2nd 3rd UW ~242.41 ~276.51 ~311.78 UV ~167 ~189.5 ~215 Almost a half are in the same quartile (47%).

50

Equations Relating Whole blood and DBS values and other assays: Total Cholesterol. Chol tot y a b x R2 N VT Serum 78.8828 +0.6351 Heritage DBS 0.30 86 VT Serum 54.4861 +0.4972 UW chol DBS 0.55 90 VT Serum 63.00789 +0.786572 UW chol eq 0.55 90 Heritage DBS 101.84 +0.2769 UW cholDBS 0.23 86 Heritage DBS 106.5818 +0.438086 UW chol eq 0.23 86

The R2 indicates that the University of Washington values are more highly correlated with the venous blood values (R2 .55 than the Heritage values (R2 .30) The Two DBS values are not highly related (R2.23)

51

Estimated DBS values based on regression equations and Z scores When the regression equation is used to estimate serum blood values from the DBS values, the means for the estimated DBS values are the same as those for the venous values but the SD is quite a bit lower – especially for the Heritage values. Using the estimated values the percent high is much lower in the regression estimated values. Mean SD Range Percent high Serum Blood 190.22 35.34 110.0-285.0 7.78 Estimated Serum –UWDBS 190.22 26.19 137.48-254.72

2.22

Mean SD Range Percent

high Serum Blood 191.38 35.52 110.00-285.00 8.14 Estimated Serum -Heritage DBS

191.38

19.55

151.28-256.08

1.16

We can also make the conversion by changing the DBS values to Z scores and then convert the Z score into a whole blood value using the mean and SD of the whole blood distribution. When this is done, the means and standard deviations are similar. The estimated % with high levels is higher than in the whole blood distribution but it has been substantially reduced from the original untransformed values.

Total Cholesterol Average S.D. Range #Cases %high risk (>=240)

Whole blood 190.22 35.34 110.00-285.00 90 7.78% Washington Converted to Whole blood 190.22 35.34 119.06-277.25 90

8.89% Whole blood 191.38 35.52 110.00-285.00 86 8.14% Heritage Converted to Whole blood 191.38 35.52 118.54-308.90 86

11.63%

52

The percentage with high levels of Total Cholesterol (N=90) Whole blood 7.78% DBS assay UW original 75.56% DBS used in regression est 2.22% DBS assay converted using Z scores 8.89%

(N=86) Whole blood 8.14% Heritage DBS assay original 4.56% Heritage DBS used in regression est 1.16% Heritage DBS assay converted using Z scores 11.63%

Because the association of the DBS assays and venous blood assays are relatively weak, it seems inappropriate to transform the equations using the regression approach. Making the transformation results in a reduced range and relatively small SD for the values. Transforming the values using Z scores seems more appropriate where the associations are low.

53

Figure 4. Total Cholesterol in HRS Samples and NHANES HRS Sample (2006, 2008 assayed in 2 sources – Biosafe and Univ. of Washington (called HRS 2008 DBS)), NHANES (Weighted)

The distribution for HRS assays from 2006 and 2008 – both done at Biosafe are quite different. The University of Washington asay is closer to the 2008 Biosafe assay but there are a high number of very high values – as in the validation sample.

54

Comparison of HDL Cholesterol from Dried Blood Spots and Venous Blood

Dried blood spots were assayed at

1) Heritage was assayed using heritage cards (N=90). The mean was 51.9 (range is 30-94)

2) UW assays were done from Biosafe cards (N= 91). The mean was 75.6 (range 46–112).

3) UW provides a Plasma equivalent with a mean of 44.2.

Venous blood was assayed at the University of Vermont lab - serum (N=94) - range is 24-93 mean is 53.3 Overview of Results The HDL values are similar to the Total cholesterol values in that the University of Washington assay produces values much higher than the others. On the other hand, the association between the University of Washington DBS values and the venous blood values is stronger (.48) than that of Heritage DBS (.32). The association of the two DBS assay values is low (.20). Z scores may be a better method of transformation than regression estimates of venous equivalents.

55

1. Descriptive Information on all HDL Cholesterol and Venous Blood Assays

Cholesterol- HDL Average Range #Cases Interquartile VT Serum 53.3 24-98 94 25 Heritage DBS 51.92 30-94 90 20 UW DBS 75.62 46-112 91 23 UW Plas Eq 44.2 13-82 91 23.6

2. % Adverse levels of HDL Cholesterol (<40) – with no adjustments

% N Vermont serum (N=94) 18.09 17 Heritage (N=90) 20.00 18 Washington (N=91) 0.00 0 Washington Equivalent (N=91) 39.56 36 As for total cholesterol, the values of HDL from University of Washington DBS are much higher than the venous blood or the Heritage values. The average of the Heritage values is close to the venous mean. The University of Washington Plasma Equivalent mean value is the lowest. Using cutoffs with these values shows that this is problematic. None of the University of Washington values are below the normal cutoff.

56

3. Distribution of HDL Cholesterol (%) Washington Washington

Equivalent Heritage Vermont

-30 19.78 1.11 3.19 30-33 5.49 1.11 3.19 33-36 5.49 8.89 3.19 36-39 7.69 8.89 8.51 39-42 6.59 7.78 9.57 42-45 5.49 5.56 7.45 45-48 3.30 5.49 14.44 6.38 48-51 2.20 7.69 8.89 9.57 51-54 1.10 8.79 7.78 3.19 54-57 1.10 10.99 6.67 9.57 57-60 7.69 4.40 3.33 2.13 60-63 8.79 2.20 6.67 7.45 63-66 4.40 2.20 4.44 4.26 66-69 4.40 3.30 3.33 8.51 69-72 7.69 1.10 3.33 6.38 72-75 6.59 1.10 2.22 0.00 75-78 6.59 0.00 0.00 4.26 78-81 6.59 1.10 3.33 0.00 81-84 8.79 1.10 1.11 1.06 84-87 9.89 0.00 0.00 0.00 87-90 6.59 0.00 0.00 1.06 90+ 14.29 0.00 1.11 1.06 The University of Washington has a substantial number of high values.

57

4. Descriptives Matched samples for HDL Cholesterol

HDL Cholesterol Average S.D. Range #Cases Correlation

Coeff. VT Serum 52.85 14.65 24-98 89 0.56177

<.0001 DBS Heritage 51.81 13.23 30-94 89 0.4542 VT Serum 53.04 14.47 24-98 90 0.69522

<.0001 DBS UW 75.78 14.38 45.91-111.72 90 <.0001 VT Serum 53.04 14.47 24-98 90 0.69522

<.0001 DBS UWPlaEQ 44.37 14.90 13.43-81.59 90 <.0001 DBS Heritage 52.07 13.21 30-94 89 0.45030 DBS UW 75.63 14.53 45.91-111.72 89 <.0001

The differences are similar to those described above.

58

Figure 1. Scatter Plots of the Validation Samples

Vermont: HDL (mg/dL)-Serum

0

10

20

30

40

50

60

70

80

90

100

Heritage lab: HDL

0 10 20 30 40 50 60 70 80 90 100

Heritage against Vermont

Number of Observations Read 95 Number of Observations Used 89 Number of Observations with Missing Values 6 R-Square 0.3156 Serum (uvhdl) = 20.63492 + 0.621881* Heritage DBS (hdbhdl)

59

Vermont: HDL (mg/dL)-Serum

0

20

40

60

80

100

120

140

UW: HDL (mg/dL)

0 20 40 60 80 100 120 140

Washington against Vermont

Number of Observations Read 95 Number of Observations Used 90 Number of Observations with Missing Values 5 R-Square 0.4833 Serum (uvhdl) = 0.042007 + 0.699461* Univ of Washington DBS (uwhdl)

60

Vermont: HDL (mg/dL)-Serum

0

20

40

60

80

100

120

140

UW: HDL plasma equivalent conc (mg/dL)

0 20 40 60 80 100 120 140

Washington Equivalent against Vermont

Number of Observations Read 95 Number of Observations Used 90 Number of Observations with Missing Values 5 R-Square 0.4833 Seru (uvhdl) = 23.08305 + 0.675324*Univ of Washington DBS (uwhdleq)

61

Heritage lab: HDL

0

20

40

60

80

100

120

140

UW: HDL plasma equivalent conc (mg/dL)

0 20 40 60 80 100 120 140

Washington Equivalent against Heritage

Number of Observations Read 95 Number of Observations Used 89 Number of Observations with Missing Values 6 R-Square 0.2028 Heritage DBS (hdbhdl) = 34.60038 + 0.395064* Univ of Washington DBS (uwhdleq)

62

Heritage lab: HDL

0

20

40

60

80

100

120

140

UW: HDL (mg/dL)

0 20 40 60 80 100 120 140

Washington against Heritage

Number of Observations Read 95 Number of Observations Used 89 Number of Observations with Missing Values 6 R-Square 0.2028 Heritage DBS (hdbhdl) = 21.12137 + 0.409185* Univ of Washington DBS (uwhdl)

63

Figure 2. HDL Bland Altman Plots Total Cholesterol Difference between Heritage DBS HDL and U Vermont Serum Average = (HeritageDBS + UVhdlserum)/2 Difference = HeritageDBS - UVhdlserum

Difference

-35

-25

-15

-5

5

15

25

35

Average25 35 45 55 65 75 85

HDL

Mean 95% CI Average 52.33 49.77 - 54.89 Heritage – Vermont Serum -1.04 -3.76 – 1.68 # of out of range (2SD): 5 Somewhat larger spread at higher levels.

64

Difference between UWashington DBS HDL and U Vermont Serum Average = (UWhdlDBS + UVhdlserum)/2 Difference = UWhdlDBS - UVhdlserum

Mean 95% CI Average 64.41 61.67 – 67.15 UWash – Vermont Serum 22.73 20.4 – 25.06 # of out of range (2SD): 5 Somewhat less precision at higher levels.

65

Figure 3. Differences Between DBS value and Vermont Serum by level of Vermont Serum

HDL Difference

-60

-40

-20

0

20

40

60

0 20 40 60 80 100

Vermont (UV)

HD

L D

iff

UV-HeritageUV-Washington UV-WashingtonEq

66

Quartiles for UWhdlDBS and UVhdlserum (N=89) Heritage uv 1st qt 2nd qt 3rd qt 4th qt 1st qt 8.99

(N=8) 12.36

(N=11) 5.62

(N=5) 0.0

(N=0) 2nd qt 7.87

(N=7) 10.11 (N=9)

3.37 (N=3)

4.49 (N=4)

3rd qt 5.62 (N=5)

2.25 (N=2)

10.11 (N=9)

4.49 (N=4)

4th qt 3.37 (N=3)

3.37 (N=3)

2.25 (N=2)

15.73 (N=14)

Cutpoints for Quartiles 1st 2nd 3rd Heritage ~41.00 ~50.00 ~61.00 UV ~41.00 ~51.00 ~63.00 45% are in the same quartile. Quartiles for UWhdlDBS and UVhdlserum (N=90) Uw uv 1st qt 2nd qt 3rd qt 4th qt 1st qt 16.67

(N=15) 7.78

(N=7) 2.22

(N=2) 0.0

(N=0) 2nd qt 6.67

(N=6) 12.22

(N=11) 5.56

(N=5) 1.11

(N=1) 3rd qt 1.11

(N=1) 3.33

(N=3) 11.11

(N=10) 7.78

(N=7) 4th qt 1.11

(N=1) 1.11

(N=1) 6.67

(N=6) 15.56

(N=14) Cutpoints for Quartiles 1st 2nd 3rd UW ~65.02 ~76.44 ~86.27 UV ~41.00 ~51.00 ~63.00 56% are in the same quartile.

67

Equations Linking Venous Blood and DBS Assays and other Assays HDL Equa y a b x R2 N VT Serum 20.63492 +0.62188 Heritage 0.32 89 VT Serum 0.042 +0.69946 UW HDL 0.48 90 VT Serum 23.08305 +0.675324 UW HDL eq 0.48 90 Heritage DBS 21.1214 +0.4092 UW HDL 0.2 89 Heritage DBS 34.60038 +0.385064 UW HDL eq 0.2 89

The association between the University of Washington DBS values and the venous blood values is stronger (.48) than that of Heritage DBS (.32). The association of the two DBS assay values is low (.20).

68

Estimated DBS values bassed on regression equations and Z scores When DBS values are used with the regression equations to estimate venous equivalents, the means for both the University of Washington and the Heritage samples are the same as that for the venous sample but the SDs are smaller and the range is narrower. The percent with adverse levels is twice as high in the whole blood sample as the University of Washington value based on DBS. The Heritage percent high is very low relative to that from comparable venous assays.

Mean SD Range

Percent Adverse levels

Serum Blood 52.85 14.65 24.00-98.00 19.10% Estimated Serum -Heritage DBS 52.85 8.23 39.29-79.09

2.25%

Mean SD Range Percent high Serum Blood 53.04 14.47 24.00-98.00 17.78% Estimated Serum –UWDBS 53.04 10.06 32.15-78.18

8.89%

We can also make the conversion by changing the DBS values to Z scores and then convert the Z score into a whole blood value using the mean and SD of the distribution. When this is done, the means and SDs for the DBS and the venous blood are essentially the same. The estimated % with adverse levels is higher in the estimated values from the University of Washington DBS and the Heritage DBS than in venous blood.

HDL Cholesterol Average S.D. #Cases Range %adverse

levels (<40) Whole blood 53.04 14.47 90 24.00-98.00 17.78% Washington Converted to Whole blood 53.04 14.47 90 22.99-89.20

22.22% Whole blood 52.85 14.65 89 24.00-98.00 19.10% Heritage Converted to Whole blood 52.85 14.65 89 28.71-99.56

21.35%

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The percentage with Low levels of HDL Cholesterol (N=90) Whole blood 17.78% UW DBS assay original 0% UW DBS used in regression est 8.89% UW DBS assay converted using Z scores 22.22% (N=89) Whole blood 19.10% Heritage DBS assay original 20.22% Heritage DBS used in regression est 2.25% Heritage DBS assay converted using Z scores 21.35%

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Figure 4. HDL Cholesterol in HRS Samples and NHANES HRS Sample (2006, 2008 assayed in 2 sources – Biosafe and Univ. of Washington, NHANES (Weighted)

The University of Washington distribution looks very different from the Biosafe distributions for 2006 and 2008. HRS2006 is very similar to NHANES. The values for 2008 are lower.

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Comparison of Cystatin C DBS and Venous Assays General Information Assays were done at Vermont and the University of Washington. Two DBS cards (one is the 2010 Heritage Card sent from HRS and the other is a Biosafe card used by HRS in 2006 and 2008 sent frozen from UCLA) and one whole blood serum sample were done at the University of Vermont.. The differences in the assays on the two types of cards represent differences in both handling and card type. From the validation sample of 82 people we provide direct comparisons of assays from whole blood and DBS both done at the University of Vermont. A DBS assay was also done a the University of Washington in the Summer of 2012. Spots were storedat -70 degrees until the assay had been developed and validated. This assay was done in duplicate so two values are provided. The N is lower for this assay because these assays were not originally planned and they were done from extra sample on spots. Overview of Results The mean of the serum values is considerably higher than that of the Vermont DBS assays. The Univ of Washington DBS values are closer to the Serum value – one is almost exacty the same. However, the serum values for the Vermont assay do not appear to be on the same scale as those in the literature. The Univ of Wash plasma equivalent value is considerably higher than than the Vermont Serum value. The correlation of the serum and DBS values is fairly high (~.76 - .81). The correlation between the DBS assays based on the two cards both done at the Univ of Vermont is even higher (.90) and the means are almost identical; the strongest association is between the two assays done at the UW (.96). The UW DBS assay is more strongly related to the Biosafe VT than the Heritage VT assay. The somewhat smaller value for the Heritage DBS card could be related to either the handling of the sample or to the card itself. The serum value from the Univ of Vermont assay does not have the same range as Cystatin C assays in the literature. It has much lower values and conventional cutoffs for high risk cannot be used. The moderately strong association between the DBS and serum values means that we could use either the regression or the Z score approach to get a value very close to the value produced by the Serum CRP assay; however, because this assay value is quite different from those reported in the literature, the value of doing this is not clear. The distribution of Cystatin C values in HRS is almost the same in 2006 and 2008 and very different from NHANES.

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1. Descriptives of All Assay Results – Univ of Vermont and University

of Washington– (Heritage and Biosafe refer to types of cards Assayed at the Univ of VT in this section)

Average Range #Cases Interquartile

Range

DBS Heritage-Assay VT 0.52 0.294-1.05 87 0.168

DBS Biosafe-Assay Vt 0.56 0.336-1.848 83 0.126

VT Serum 0.749 0.41-1.39 94 0.21

DBS Washington 1 0.668 0.480-1.473 34 0.111

DBS Washington 2 0.751 0.516-1.563 34 0.171

UW PlasmaEqui 0.965 0.715-1.936 34 0.163

2. Percent high risk Cystatin C with traditional risk factor definition.

%High Cystatin C (>1.55 mg/l) % N

Heritage Card (N=87) 0.0 0

Biosafe Card (N=83) 1.2 1

Serum (N=94) 0.0 0

Washington 1 (N=34) 0.0 0

Washington 2 (N=34) 2.94 1

Washington Plasma Equi (N=34)

2.94 1

None of the assays produce values in the range traditionally used to designate high risk.

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3. Distribution of Cystatin C (%)

Serum Heritage Biosafe Washington 1

Washington 2

Washington Plasma equi

<0.3 0.00 1.15 0.00 0.00 0.00 0.00

0.3-0.4 0.00 16.09 9.64 0.00 0.00 0.00

0.4-0.5 4.26 33.33 30.12 11.76 0.00 0.00

0.5-0.6 10.64 32.18 38.55 32.35 14.71 0.00

0.6-0.7 30.85 9.20 7.23 35.29 32.35 0.00

0.7-0.8 25.53 2.30 8.43 5.88 23.53 17.65

0.8-0.9 12.77 4.60 1.20 5.88 17.65 26.47

0.9-1.0 7.45 0.00 1.20 5.88 2.94 26.47

1.0-1.1 2.13 1.15 2.41 0.00 5.88 14.71

1.1-1.2 4.26 0.00 0.00 0.00 0.00 2.94

1.2+ 2.13 0.00 1.20 2.94 2.94 11.76

The serum values for Vermont as well as the DBS values seem to be on a different scale from more commonly done assays reported in the literature.

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4. Equivalent Sample comparisons: Cystatin C

Cystatin C Average S.D. Range #Cases Correlation

Coeff. Serum 0.74 0.17 0.41-1.22 86 0.80858

<.0001 DBS Heritage 0.52 0.14 0.29-1.05 86 <.0001 Serum 0.75 0.18 0.41-1.39 82 0.78319

<.0001 DBS Biosafe 0.56 0.20 0.34-1.85 82 <.0001 DBS Heritage VT 0.52 0.14 0.29-1.05 78 0.89918 DBS Biosafe VT 0.54 0.15 0.34-1.05 78 <.0001 <.0001 Serum 0.71 0.18 0.41-1.39 33 0.75722 DBS Washington1 0.67 0.19 0.52-1.56 33 <.0001 <.0001 Serum 0.71 0.18 0.41-1.39 33 0.79537 DBS Washington2 0.75 0.20 0.52-1.56 33 <.0001 <.0001 DBS Heritage 0.51 0.11 0.38-0.84 31 0.72522 DBS Washington1 0.64 0.13 0.48-0.98 31 <.0001 <.0001 DBS Heritage 0.51 0.11 0.38-0.84 31 0.78268 DBS Washington2 0.72 0.13 0.52-1.10 31 <.0001 <.0001 DBS Biosafe 0.57 0.26 0.34-1.85 34 0.90118 DBS Washington1 0.67 0.19 0.48-1.47 34 <.0001 <.0001 DBS Biosafe 0.57 0.26 0.34-1.85 34 0.90975 DBS Washington2 0.75 0.19 0.52-1.56 34 <.0001 <.0001 DBS Washington1 0.67 0.19 0.48-1.47 34 0.95785 DBS Washington2 0.75 0.19 0.52-1.56 34 <.0001 <.0001

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Figure 1. Scatter Plots of the Validation Sample Two DBS Assays

Vermont: Cystatin C - Biosafe (mg/L)

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

Vermont: Cystatin C - Heritage (mg/L)

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1

Heritage against Biosafe

Number of Observations Read 95 Number of Observations Used 78 Number of Observations with Missing Values 17 R-Square 0.8085 Vermont DBS Biosafe Card (uvcycdb2)= 0.05702 + 0.94184* Vermont DBS Heritage Card (uvcycdb1);

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DBS_CYSC2

0

1

2

DBS_CYSC1

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

DBS Washington2 against DBS WAshington1

Number of Observations Read 95 Number of Observations Used 34 Number of Observations with Missing Values 61 R-Square 0.9175 DBS Washington 2 (dbs_cysc2)= 0.09350 + 0.98483* DBS Washington 1 (dbs_cysc1);

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Heritage against Serum Vermont: Cystatin C - Serum (ug/mL)

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

Vermont: Cystatin C - Heritage (mg/L)

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1

Number of Observations Read 95 Number of Observations Used 86 Number of Observations with Missing Values 9 R-Square 0.6538 Serum (uvcycsrm) = 0.22910 + 0.409185* DBS Heritage(uvcycdb1)

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Vermont: Cystatin C - Serum (ug/mL)

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

Vermont: Cystatin C - Biosafe (mg/L)

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9

Biosafe against Serum

Number of Observations Read 95 Number of Observations Used 82 Number of Observations with Missing Values 13 R-Square 0.6134 Serum (uvcycsrm) = 0.35451 + 0.69954* DBS Vt Bio (uvcycdb2)

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Vermont: Cystatin C - Serum (ug/mL)

0

1

2

DBS_CYSC1

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

DBS Washington1 against Serum

Number of Observations Read 95 Number of Observations Used 33 Number of Observations with Missing Values 62 R-Square: 0.5734 Serum (uvcycsrm) = 0.222746 + 0.730338*DBS Washington1 (DBS_CYSC1)

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Vermont: Cystatin C - Serum (ug/mL)

0

1

2

DBS_CYSC2

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

DBS Washington2 against Serum

Number of Observations Read 95 Number of Observations Used 33 Number of Observations with Missing Values 62 R-Square: 0.6326 Serum (uvcycsrm) = 0.147552 + 0.748093 * DBS Washington2(DBS_CYSC2)

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Vermont: Cystatin C - Serum (ug/mL)

0

1

2

DBS_CYSC2

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

DBS Washington2 against Serum

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Figure 2. Cystatin C Bland Altman Plots Difference between Heritage DBS and Serum Average = (Heritage DBS + Serum)/2 Difference = Heritage DBS – Serum

Difference

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

Average0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2

Cystatin C (Serum and Heritage)

Mean 95% CI Average 0.628 0.60 - 0.66 Heritage – Serum -0.224 -0.24 - -0.20 # of out of range (2SD): 3

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Difference between Biosafe DBS and Serum Average = (Biosafe DBS + Serum)/2 Difference = Biosafe DBS – Serum

Difference

-0.5-0.4-0.3-0.2-0.10.00.10.20.30.40.5

Average0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7

Cystatin C (Serum and Biosafe)

Mean 95% CI Average 0.655 0.62 – 0.70 Biosafe - Serum -0.186 -0.22 - -0.16

# of out of range (2SD): 3

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Difference between DBS Washington1 and Serum Average = (DBS Washington1 + Serum)/2 Difference = DBS Washington1 – Serum

Cystatin C (Serum and DBS Washington 1)

Difference

-0.35

-0.15

0.05

0.25

0.45

Average0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

HBA1C

Bland-Altman Plot

Mean 95% CI Average 0.691 0.35 – 1.03 DBS Washington1 – Serum -0.042 -0.30 – 0.21 # of out of range (2SD): 2

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Difference between DBS Washington2 and Serum Average = (DBS Washington2 + Serum)/2 Difference = DBS Washington2 – Serum

Difference

-0.3

-0.1

0.1

0.3

0.5

Average0.5 0.7 0.9 1.1 1.3 1.5

Cystatin C

Bland-Altman Plot

Mean 95% CI Average 0.733 0.38 – 1.09 DBS Washington2 - Serum 0.042 -0.20 - 0.28

# of out of range (2SD): 1

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Figure 3. Differences Between Assays by Level of Venous Assay

Cystatin C Difference (Serum - Heritage)

-0.6-0.5-0.4-0.3-0.2-0.1

00.10.20.3

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Serum Cystatin C

Cyc

Diff

Cystatin C Difference (Serum - Biosafe)

-0.5-0.4-0.3-0.2-0.1

00.10.20.30.40.5

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Serum Cystatin C

Cyc

Diff

87

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Quartiles for Cystatin C – Heritage DBS versus Serum (N=86) serum heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 12.79 (N=11)

10.47 (N=9)

2.33 (N=2)

1.16 (N=1)

2nd qt 1.16 (N=1)

11.63 (N=10)

8.14 (N=7)

0.0 (N=0)

3rd qt 1.16 (N=1)

9.30 (N=8)

9.30 (N=8)

6.98 (N=6)

4th qt 1.16 (N=1)

2.33 (N=2)

2.33 (N=2)

19.77 (N=17)

53% are in the same quartile on the two assays. Cutpoints for Quartiles 1st 2nd 3rd Heritage DBS ~0.42 ~0.483 ~0.588

Vermont serum ~0.62 ~0.70 ~0.82

Quartiles for Cystatin C – Biosafe DBS versus Serum (N=82) serum biosafe

1st qt 2nd qt 3rd qt 4th qt

1st qt 21.95 (N=18)

1.22 (N=1)

1.22 (N=1)

1.22 (N=1)

2nd qt 12.20 (N=10)

7.32 (N=6)

3.66 (N=3)

1.22 (N=1)

3rd qt 3.66 (N=3)

2.44 (N=2)

8.54 (N=7)

9.76 (N=8)

4th qt 1.22 (N=1)

1.22 (N=1)

2.44 (N=2)

20.73 (N=17)

59% are in the same quartile of the two assays. Cutpoints for Quartiles 1st 2nd 3rd Biosafe DBS ~0.462 ~0.504 ~0.588

Vermont serum ~0.62 ~0.705 ~0.820

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Quartiles for Cystatin C – DBS Washington 1 versus Serum (N=) serum Washington1

1st qt 2nd qt 3rd qt 4th qt

1st qt 18.18 (N=6)

3.03 (N=1)

3.03 (N=1)

3.03 (N=1)

2nd qt 3.03 (N=1)

15.15 (N=5)

6.06 (N=2)

0.00 (N=0)

3rd qt 6.06 (N=2)

0.00 (N=0)

9.09 (N=3)

9.09 (N=3)

4th qt 3.03 (N=1)

3.03 (N=1)

6.06 (N=2)

12.12 (N=4)

55% are in the same quartile on both assays. Cutpoints for Quartiles 1st 2nd 3rd DBS Washington1

~0.580 ~0.622 ~0.691

Vermont Serum ~0.62 ~0.68 ~0.79

Quartiles for Cystatin C – DBS Washington 2 versus Serum (N=) Serum Washington2

1st qt 2nd qt 3rd qt 4th qt

1st qt 18.18 (N=6)

6.06 (N=2)

0.00 (N=0)

3.03 (N=1)

2nd qt 6.06 (N=2)

9.09 (N=3)

9.09 (N=3)

0.00 (N=0)

3rd qt 3.03 (N=1)

6.06 (N=2)

12.12 (N=4)

3.03 (N=1)

4th qt 3.03 (N=1)

0.00 (N=0)

3.03 (N=1)

18.18 (N=6)

58% are in the same quartile on both assays. Cutpoints for Quartiles 1st 2nd 3rd DBS Washington2

~0.645 ~0.718 ~0.816

Vermont serum ~0.62 ~0.68 ~0.79

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Equations Linking Serum and DBS for Cystatin C

Cystatin C a B X R2 N

Serum 0.22910 +0.98922 DBS Heritage 0.65 86

Serum 0.35451 +0.69954 DBS Biosafe 0.61 82

Equations with cases deleted When the high value (on Biosafe card) that is an outlier is deleted, the equation is A= 0.05379, b=.66548, and R2= .60 When the other outlier is removed the values are A= .00347, b=.72663, and R2=.69 Cystatin C a B X R2 N

Serum 0.222746 +0.730338 DBS Washington1 0.57 33

Serum 0.147552 + 0.748093 DBS Washington2 0.63 33

Equations Linking DBS Cystatin C y a B X R2 N

Heritage VT 0.05014 0.85844 Biosafe 0.8085 78

Biosafe VT 0.05702 0.94184 Heritage 0.8085 78

Washington1 0.15097 0.96865 Heritage 0.6715 31

Washington1 0.28756 0.66499 Biosafe 0.8389 34

Washington2 0.15807 1.11569 Heritage 0.8110 31

Washington2 0.35482 0.69315 Biosafe 0.8622 34

Heritage VT 0.06138 0.69319 Washington1 0.6715 31

Heritage VT -0.01937 0.72688 Washington2 0.8110 31

Biosafe VT -0.27060 1.26149 Washington1 0.8389 34

Biosafe VT -0.36251 1.24384 Washington2 0.8622 34

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Cystatin C y a B x R2 N

Washington1 -0.03198 0.93160 Washington2 0.9175 34

Washington2 0.09350 0.98483 Washington1 0.9175 34

Estimated DBS Values of Cystatin C from Regression and Z scores (outliers deleted) It is possible to use these regression equations to estimate whole blood values based on the DBS value. When this is done, using the Serum-2 equation above, the means of the whole blood and the estimated serum from DBS are the same and the SD is smaller and the percent with high levels is twice as high in the whole blood sample. Mean SD Range Percent high Whole Blood 0.74 0.17 0.41-1.22 0% Estimated Whole blood - Heritage DBS 0.75 0.18 0.46-1.44

0%

Mean SD Range Percent high Whole Blood 0.74 0.16 0.47-1.22 0% Estimated Whole blood – Biosafe DBS 0.74 0.14 0.55-1.23

0%

Mean SD Range Percent high Whole Blood 0.71 0.18 0.41-1.39 0% Estimated Whole blood –DBS Washington1 0.71 0.14 0.57-1.30

0%

Mean SD Range Percent high Whole Blood 0.71 0.18 0.41-1.39 0% Estimated Whole blood –DBS Washington2 0.71 0.15 0.53-1.32

0%

We can also make the conversion by changing the DBS values to Z scores and then convert the Z score into a whole blood value using the mean and SD of the distribution. When this is done, the means and SDs are essentially the same. High values of Cystatin C indicate poor kidney functioning (>1.55 is the

92

conventional cutoff). No one reaches these values in either the DBS or the serum assays done at the University of Vermont.

Cystatin C Average S.D. #Cases Range %high risk

(>1.55) Whole blood 0.74 0.17 86 0.41-1.22 0% Heritage Converted to Whole blood 0.74 0.17 86 0.47-1.39

0% Whole blood 0.75 0.18 82 0.41-1.39 0% Biosafe Converted to Whole blood 0.75 0.18 82 0.55-1.90

0%

Cystatin C Average S.D. #Cases Range %high risk

(>1.55) Whole blood 0.71 0.18 33 0.41-1.39 0% Washington1 Converted to Whole blood 0.71 0.18 33 0.53-1.49

0% Whole blood 0.71 0.18 33 0.41-1.39 0% Washington2 Converted to Whole blood 0.71 0.18 33 0.49-1.47

3.03%

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The percentage with High Values of Cystatin C

(N=80) Whole blood 0% Biosafe DBS assay original 0% Biosafe DBS used in regression est 0% Biosafe DBS assay converted using Z scores 0% (n=80) Whole blood 0% Heritage DBS assay original 0% Heritage DBS used in regression est 0% Heritage DBS assay converted using Z scores 0%

(N=33) Whole blood 0% Biosafe DBS assay original 0% Biosafe DBS used in regression est 0% Biosafe DBS assay converted using Z scores 0% (n=33) Whole blood 0% Heritage DBS assay original 3.03% Heritage DBS used in regression est 0% Heritage DBS assay converted using Z scores 0%

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Figure 4. Cystatin C in HRS Samples and NHANES (1999-2002)

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Comparisons of CRP DBS and Venous Blood Assays General Information Dried Blood Spots were collected on two types of cards and assayed at three labs.

1) 2010 Heritage cards sent from HRS and assayed in Vermont (N=83) 2) 2006-2008 Biosafe cards sent frozen from UCLA and assayed in Vermont

(N=79) 3) 2006-2008 Biosafe cards assayed by Thom McDade (N=64) 4) Cards sent frozen from UCLA to the University of Washington

Whole blood was separated and frozen and sent to the University of Vermont lab – Serum (N=91).

From the validation sample, we provide direct comparisons of the values from the DBS assays and the serum assays both done at the University of Vermont. One issue that must be addressed before comparing the samples is that the lower detectible limit of the DBS assays done in Vermont is .63 and about half of the assays are in the undetectable range. We impute values to the nondetectible cases- .428 for the DBS - which is the average value of a set of cases that were assayed by hand from surplus HRS DBS. For the actual HRS data, assays were redone for DBS with low values. The lower detection limit for the serum assay is .16 and 3 cases are below this level. For the serum assay, we assign .10 to the nondetectable cases. We examine descriptive statistics with and without these imputed cases in the sample. The means and SD of the serum samples are much higher than those of the DBS sample when the imputed values are included for cases below detection. The initial University of Washington assay does not include values above 10.75. In the second assay, samples that were above detection (coded OOR-high) produced values 21.59, 29.24, 78.32). Overview of Results The association between the Vermont DBS assays and the venous blood assays are very high. The correlation coefficients between the venous blood and the DBS values are .98-.99. The UW2 DBS assay is also relatively highly correlated with the serum levels (.86) and this value is similar for the MdDade assay (.89). The UW1 assay with the reduced range is not hihgly related to serum values (.56). The differences between the DBS and serum values are larger at higher levels. When both values are log transformed, as is often the case in CRP analysis, the differences are similar across the assay range.

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The McDade DBS assay, the Vermont DBS assay, and the Washington assays have very different values and ranges. McDade’s assay detects much lower values but it has a severely restricted range at the top. The correlations between the sets of DBS assays are generally high with the exception of the Washington 1 assay and the McDade and Washington 2 assay. The values of the Vermont serum assays differ from other serum assays of CRP; they have much lower values.

Because the relationships among these assays are so high, various approaches to converting them into similar metrics would work reasonably well. The distribution of the CRP values in HRS in 2006 and 2008 is very similar. The values are lower than those in NHANES and similar to those in NSHAP.

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1. Descriptives for the DBS and Venous Blood Assays for CRP

CRP Average Range #Cases Interquartile Range VT Serum 2.87 0.16-43.8 91 2.48 DBS Her Vt 3.05 0.63-18.44 30 2.31 DBS Bio VT 3.86 0.63-38.14 42 2.23 DBS McDade 1.95 0.02-17.60 64 1.28 DBS Washington1 2.28 0.076-10.752 52 2.64 DBS Washington 2 4.36 0.096-78.32 55 3.02 PlasmaEqui

Descriptives for the Cases with Values from both DBS and Venous CRP

CRP Average Range #Cases S.D. Correlation Coeff.

VT Serum 7.12 1.51-43.80 30 8.66 0.98188 <.0001 DBS Her VT 3.05 0.63-18.44 30 3.70

0.0001 VT Serum 5.33 0.45-43.80 41 7.79 0.98804

<.0001 DBS Bio VT 3.93 0.63-38.14 41 6.34 <.0001 VT Serum 3.15 0.16-43.80 60 6.62 0.88526 McDade 2.07 0.05-17.60 60 3.81 <.0001 0.0273 VT Serum 1.93 0.16-19.70 49 3.06 0.56088 DBS Washington1 2.38 0.37-10.75 49 2.37 <.0001 <.0001 VT Serum 3.06 0.16-43.80 52 6.89 0.87086 DBS Washington2 4.58 0.34-78.32 52 11.56 <.0001 .0024 DBS Her VT 3.43 0.63-18.44 20 4.10 0.80184 McDade 5.16 0.99-17.60 20 5.39 <.0001 .0014 DBS Bio VT 4.47 0.63-38.14 27 7.49 0.81560 McDade 3.70 0.47-17.60 27 4.76 <.0001 0.0046 DBS Her VT 2.03 0.63-7.94 15 1.84 0.14734 DBS Washington1 4.65 2.36-10.75 15 2.08 0.6003 0.0008 DBS Bio VT 2.40 0.63-13.82 26 2.73 0.46724 DBS Washington1 3.81 1.49-10.75 26 2.47 0.0161

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0.0001 McDade 1.36 0.02-17.60 44 2.83 0.41938 DBS Washington1 2.25 0.08-10.75 44 2.48 0.0046 0.0026 DBS Her VT 3.19 0.63-18.44 18 4.39 0.84391 DBS Washington2 10.78 2.28-78.32 18 18.30 <.0001 0.0067 DBS Bio VT 3.96 0.63-38.14 29 7.31 0.89091 DBS Washington2 7.61 1.46-78.32 29 14.89 <.0001 0.0069 McDade 1.96 0.02-17.60 47 3.93 0.63843 DBS Washington2 4.70 0.10-78.32 47 12.17 <.0001 0.0013

Descriptives when Vermont DBS that is below the detectible limit is assigned .428 for <.63

CRP Average Range #Cases S.D. Correlation Coeff.

VT Serum 3.01 0.16-43.80 83 6.03 0.98523 <.0001 DBS Her VT 1.38 0.428-18.44 83 2.54

<.0001 VT Serum 3.02 0.16-43.80 79 6.08 0.98854

<.0001 DBS Bio VT 2.25 0.428-38.14 79 4.87 <.0001 VT Serum 3.15 0.16-43.80 60 6.62 0.88526 McDade 2.07 0.05-17.60 60 3.81 <.0001 .0273 VT Serum 1.93 0.16-19.70 49 3.06 0.56088 DBS Washington 1 2.38 0.37-10.75 49 2.37 <.0001 <.0001 VT Serum 3.06 0.16-43.80 52 6.89 0.87086 DBS Washington 2 4.58 0.34-78.32 52 11.56 <.0001 .0024 McDade 2.02 0.02-17.60 59 3.84 0.85812 DBS Her VT 1.45 0.428-18.44 59 2.75 <.0001 .0002 McDade 2.34 0.02-17.60 57 3.67 0.84183 DBS Bio VT 1.90 0.428-38.14 57 5.49 <.0001 .0002

99

DBS Washington1 2.18 0.08-10.75 48 2.10 0.85897 DBS Her VT 0.78 0.428-3.86 48 0.71 <.0001 <.0001 DBS Wasington2 4.47 0.096-78.32 51 11.67 .98394 DBS Her VT 1.40 0.428-18.44 51 2.88 <.0001 .0086 DBS Washington1 2.30 0.076-10.75 51 2.36 0.97782 DBS Bio VT 1.20 0.428-6.13 51 1.26 <.0001 <.0001 DBS Washington2 4.42 0.096-78.32 54 11.37 0.99678 DBS Bio VT 2.32 0.428-38.14 54 5.60 <.0001 .0061

DBS assigned .428 for <.63 (-333), Serum assigned .10 for (-333 for 3 cases)

CRP Average S.D. Range #Cases Correlation

Coeff. VT Serum 2.91 5.94 0.10-43.80 86 0.98511

<.0001 DBS Her VT 1.34 2.50 0.428-18.44 86 <.0001 VT Serum 2.91 6.00 0.10-43.80 82 0.98841 DBS Bio VT 2.18 4.79 0.428-38.14 82 <.0001 <.0001 VT Serum 3.01 6.49 0.10-43.80 63 0.88650 McDade 1.97 3.74 0.02-17.60 63 <.0001 0.0270 VT Serum 1.86 3.02 0.10-19.70 51 0.56934 DBS Washington 1 2.29 2.36 0.076-10.752 51 <.0001 <.0001 VT Serum 2.95 6.78 0.10-43.80 54 0.87163 DBS Washington 2 4.41 11.37 0.096-78.32 54 <.0001 0.0024

100

2. %High CRP with no adjustment (>= 3 ug/ml) % N Vermont (N=91) 23.08 21 Heritge DBS (N=30) 26.67 8 Biosafe (N=42) 33.33 14 McDade (N=64) 17.19 11 Washington 1 (N=52) 26.92 14 Washington 2 (N=55) 29.09 16 Washington Plasma Equi (N=55)

25.45 14

101

2. Distribution of CRP (%)

Vermont Serum

Heritage DBS - Vt

Biosafe DBS-VT

McDade Washington 1

Washington 2

Washington Plasma equi

-0.2 4.40 0.00 0.00 21.88 3.85 3.64 3.64 0.2-0.4 13.19 0.00 0.00 14.06 3.85 3.64 5.45 0.4-0.6 15.38 0.00 0.00 14.06 19.23 16.36 18.18 0.6-0.8 8.79 6.67 9.52 10.94 1.92 5.45 5.45 0.8-1.0 6.59 10.00 16.67 1.56 7.69 9.09 9.09 1.0-1.2 9.89 10.00 4.76 4.69 7.69 3.64 3.64 1.2-1.4 4.40 10.00 4.76 4.69 3.85 3.64 5.45 1.4-1.6 5.49 6.67 2.38 4.69 3.85 3.64 5.45 1.6-1.8 1.10 10.00 0.00 3.13 5.77 5.45 5.45 1.8-2.0 0.00 13.33 9.52 1.56 1.92 5.45 5.45 2.0-2.2 1.10 3.33 4.76 0.00 5.77 5.45 0.00 2.2-2.4 1.10 0.00 7.14 1.56 3.85 1.82 1.82 2.4-2.6 1.10 0.00 0.00 0.00 1.92 1.82 1.82 2.6-2.8 0.00 0.00 2.38 0.00 1.92 0.00 0.00 2.8-3.0 4.40 3.33 4.76 0.00 0.00 1.82 3.64 3.0-3.2 0.00 0.00 9.52 1.56 1.92 0.00 0.00 3.2-3.4 2.20 0.00 2.38 1.56 1.92 1.82 3.64 3.4-3.6 1.10 3.33 0.00 1.56 1.92 1.82 1.82 3.6-3.8 1.10 3.33 0.00 0.00 0.00 3.64 3.64 3.8-4.0 2.20 3.33 0.00 0.00 1.92 1.82 1.82 4.0-4.2 0.00 3.33 0.00 1.56 1.92 1.82 1.82 4.2-4.4 1.10 0.00 0.00 1.56 1.92 1.82 0.00 4.4-4.6 2.20 0.00 0.00 0.00 5.77 3.64 0.00 4.6-4.8 1.10 0.00 2.38 0.00 0.00 0.00 0.00 4.8-5.0 0.00 0.00 2.38 0.00 1.92 0.00 1.82 5.0-5.2 1.10 0.00 2.38 0.00 0.00 0.00 0.00 5.2-5.4 0.00 0.00 0.00 0.00 0.00 1.82 0.00 5.4-5.6 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5.6-5.8 1.10 0.00 2.38 0.00 0.00 0.00 1.82 5.8-6.0 0.00 0.00 0.00 1.56 0.00 0.00 0.00 6.0+ 9.89 13.33 11.90 7.81 7.69 10.91 9.09

102

Figure 1. Scatter Plots of the Validation Sample

VT Serum against Heritage Card Vermont Vermont: CRP - Serum (ug/mL)

0

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45

50

Vermont: CRP - Heritage (ug/mL)

0 5 10 15 20 25 30 35 40 45 50

g

Number of Observations Read 95 Number of Observations Used 30 Number of Observations with Missing Values 65 R-Square 0.9641 Serum(uvcrpsrm) = 0.113653 + 2.296024*DBS Heritage Card (uvcrpdb1)

103

VT Serum against Biosafe Card Vermont Vermont: CRP - Serum (ug/mL)

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45

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Vermont: CRP - Biosafe (ug/mL)

0 5 10 15 20 25 30 35 40 45 50

g

Number of Observations Read 95 Number of Observations Used 41 Number of Observations with Missing Values 54 R-Square 0.9762 Serum (uvcrpsrm) = 0.552909 + 1.2146*DBS Biosafe Card (uvcrpdb2)

104

Vermont: CRP - Serum (ug/mL)

0

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25

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35

40

45

50

McDade: CRP

0 5 10 15 20 25 30 35 40 45 50

VT serum against MCDade blood spot

Number of Observations Read 95 Number of Observations Used 60 Number of Observations with Missing Values 35 R-Square 0.7837 Serum (uvcrpsrm) = 0.389502 + 1.128949* McDade DBS (mccrp)

105

Vermont: CRP - Heritage (ug/mL)

0

2

4

6

8

10

12

14

16

18

20

Vermont: CRP - Biosafe (ug/mL)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Heritage Card Vermont against Biosafe Card Vermont

Number of Observations Read 95 Number of Observations Used 29 Number of Observations with Missing Values 66 R-Square 0.9447 DBS Heritage (uvcrpdb1) = 0.463497 + 0.506603*DBS Biosafe (uvcrpdb2)

106

Vermont: CRP - Heritage (ug/mL)

0

2

4

6

8

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12

14

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18

20

McDade: CRP

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Heritage Card Vermont against McDade blood spot

Number of Observations Read 95 Number of Observations Used 20 Number of Observations with Missing Values 75 R-Square 0.6429 DBS Vt (uvcrpdb1) = 0.28989 + 0.609481* McDade DBS (mccrp)

107

McDade: CRP

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12

14

16

18

20

Vermont: CRP - Biosafe (ug/mL)

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

McDade blood spot against Biosafe Card Vermont

Number of Observations Read 95 Number of Observations Used 27 Number of Observations with Missing Values 68 R-Square 0.6652 McDade DBS (mccrp) = 1.381059 + 0.517863* Univ Vt DBS (uvcrpdb2)

108

Vermont: CRP - Serum (ug/mL)

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2

4

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10

12

14

16

18

20

DBS_CRP1

0 1 2 3 4 5 6 7 8 9 10 11

DBS Washington1 against Serum

Number of Observations Read 95 Number of Observations Used 49 Number of Observations with Missing Values 46 R-Square: 0.3146 Serum (uvcrpsrm) = 0.207401 + 0.725995*DBS Washington1(DBS_CRP1)

109

Vermont: CRP - Serum (ug/mL)

0

5

10

15

20

25

30

35

40

45

50

DBS_CRP2

0 10 20 30 40 50 60 70 80 90

DBS Washington2 against Serum

Number of Observations Read 95 Number of Observations Used 52 Number of Observations with Missing Values 43 R-Square: 0.7584 Serum (uvcrpsrm) = 0.681604 + 0.518812 * DBS Washington2 (DBS_CRP2)

110

DBS_CRP1

0

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3

4

5

6

7

8

9

10

11

DBS_CRP2

0 1 2 3 4 5 6 7 8 9 10 11

DBS Washington2 against DBS Washington1

Number of Observations Read 95 Number of Observations Used 52 Number of Observations with Missing Values 43 R-Square 0.9915 DBS Washington1 (DBS_CRP1) = -0.132995 + 1.133832 * DBS Washington2 (DBS_CRP2)

111

McDade: CRP

0123456789

1011121314151617181920

DBS_CRP1

0 1 2 3 4 5 6 7 8 9 10 11 12

DBS Washington1 against McDade

Number of Observations Read 95 Number of Observations Used 44 Number of Observations with Missing Values 51 R-Square 0.1759 McDade (mccrp) = 0.284156 + 0.478372 * DBS Washington1 (DBS_CRP1)

112

McDade: CRP

0123456789

10111213141516171819

DBS_CRP2

0 10 20 30 40 50 60 70 80

DBS Washington2 against McDade

Number of Observations Read 95 Number of Observations Used 47 Number of Observations with Missing Values 48 R-Square: 0.4076 McDade (mccrp) = 0.989101 + 0.20642 * DBS Washington2 (DBS_CRP2)

113

Vermont: CRP - Heritage (ug/mL)

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6

7

8

9

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11

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DBS_CRP1

0 1 2 3 4 5 6 7 8 9 10 11 12

DBS Washington1 against Heritage

Number of Observations Read 95 Number of Observations Used 15 Number of Observations with Missing Values 80 R-Square 0.0217 Heritage (uvcrpdb1) = 1.427446 + 0.130118 * DBS Washington1 (DBS_CRP1).

114

Vermont: CRP - Heritage (ug/mL)

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DBS_CRP2

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

DBS Washington2 against Heritage

Number of Observations Read 95 Number of Observations Used 18 Number of Observations with Missing Values 77 R-Square 0.7122 Heritage (uvcrpdb1) = 1.009705 + 0.202247 * DBS Washington2 (DBS_CRP2)

115

Vermont: CRP - Biosafe (ug/mL)

0

12

3

45

6

7

89

10

11

1213

14

15

DBS_CRP1

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

DBS Washington1 against Biosafe

Number of Observations Read 95 Number of Observations Used 26 Number of Observations with Missing Values 69 R-Square 0.2183 Biosafe (uvcrpdb2) = 0.444671 + 0.514808 * DBS Washington1 (DBS_CRP1)

116

Vermont: CRP - Biosafe (ug/mL)

0

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25

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35

40

DBS_CRP2

0 10 20 30 40 50 60 70 80

DBS Washington2 against Biosafe

Number of Observations Read 95 Number of Observations Used 29 Number of Observations with Missing Values 66 R-Square 0.7937 Biosafe (uvcrpdb2) = 0.633131 + 0.437398 * DBS Washington2 (DBS_CRP2)

117

CRP Scatter Plots and Difference Graph (Excluding highest value 43.8 of VT serum)

VT Serum against Heritage

Vermont: CRP - Serum (ug/mL)

123456789

101112131415161718192021

Vermont: CRP - Heritage (ug/mL)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Number of Observations Read 95 Number of Observations Used 29 Number of Observations with Missing Values 66 R-Square 0.9067 Serum (uvcrpsrm) = 0.42155 + 2.1555 * DBS Heritage (uvcrpdb1)

118

VT Serum against Biosafe

Vermont: CRP - Serum (ug/mL)

0

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22

Vermont: CRP - Biosafe (ug/mL)

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Number of Observations Read 95 Number of Observations Used 40 Number of Observations with Missing Values 55 R-Square 0.9785 Biosafe DBS (uvcrpdb2) = -0.18322 + 1.47867 * Vermont Serum (uvcrpsrm)

119

Figure 2. CRP Bland Altman Plots Difference between U Vermont Serum CRP and Heritage DBP Average = (Vermont Serum + Hertitage DBS)/2 Difference = Heritage – Vermont Serum

Difference

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-25

-20

-15

-10

-5

0

5

Average0 5 10 15 20 25 30 35 40 45

CRP (with Vermont Serum and Heritage DBS)

Mean 95% CI Average 5.089 2.89-7.29 Heritage – Serum -4.071 -5.89 ~ -2.25 # of out of range (2SD): 1

120

Difference between U Vermont Serum CRP and Biosafe DBS Average = (Biosafe + Vermont Serum) /2 Difference = Biosafe – Vermont Serum

Difference

-10

-8

-6

-4

-2

0

2

4

Average0 5 10 15 20 25 30 35 40 45

CRP (with Vermont Serum and Biosafe DBS)

Mean 95% CI Average -1.397 2.47-6.79 DBS Univ VT (Biosafe) – Serum

4.632 -1.96 ~ -0.84

# of Out of range (2SD): 4

121

Difference between U Vermont Serum CRP and McDade blood spot Average = (Vermont Serum + McDade blood spot)/2 Difference = McDade blood spot – Vermont Serum

Difference

-30

-25

-20

-15

-10

-5

0

5

10

Average0 5 10 15 20 25 30

CRP (with Vermont Serum and McDade)

Mean 95% CI Average 2.611 1.33-3.89 McDade – Serum -1.082 -2.02 ~ -0.14 # of out of range (2SD): 1

122

Difference between Biosafe CRP and Heritage CRP Average = (Biosafe + Heritage)/2 Difference = Heritage – Biosafe

Difference

-20

-15

-10

-5

0

5

Average0 5 10 15 20 25 30

CRP (with Biosafe and Heritage)

Mean 95% CI Average 4.026 2.04 – 6.02 Heritage – Biosafe -2.022 -3.36 ~ -0.68 # of out of range (2SD): 1

123

Difference between McDade and Heritage CRP Average = (McDade + Heritage)/2 Difference = Heritage – McDade

Difference

-10

-5

0

5

10

Average0 5 10 15 20 25 30

CRP (with McDade and Heritage)

Mean 95% CI Average 4.082 1.87-6.29 McDade – Heritage -0.774 -2.48 – 0.94 # of out of range (2SD): 1

124

Difference between McDade and Biosafe CRP Average = (McDade + Biosafe)/2 Difference = Biosafe – McDade

Difference

-25

-20

-15

-10

-5

0

5

10

Average0 5 10 15 20 25 30

CRP (with McDade and Biosafe)

Mean 95% CI Average 4.296 2.32 – 6.28 McDade – Biosafe 1.724 0.3 – 3.14 # of out of range (2SD): 1

125

Difference between U Vermont Serum CRP and DBS Washington 1 Average = (Vermont Serum + DBS Washington1)/2 Difference = Washington1 – Vermont Serum

Difference

-5-4-3-2-10123456789

10

Average-10 -7 -4 -1 2 5 8 11 14

CRP

Bland-Altman Plot Mean 95% CI Average 2.156 1.47-2.85 Washington1 – Serum 0.444 -0.307~1.196 # of out of range (2SD): 0

126

Difference between U Vermont Serum CRP and DBS Washington 2 Average = (Vermont Serum + DBS Washington2)/2 Difference = Washington2 – Vermont Serum

Difference

-20

-15

-10

-5

0

5

10

15

20

25

30

35

40

Average0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

CRP

Bland-Altman Plot

Mean 95% CI Average 3.816 1.327-6.305 Washington2 – Serum 1.52 -0.292~3.333 # of out of range (2SD): 3

127

Difference between DBS Washington1 and DBS Washington 2 Average = (DBS Washington1 + DBS Washington2)/2 Difference = Washington1 – Washington2

Difference

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Average0 1 2 3 4 5 6 7 8 9 10 11 12

CRP

Bland-Altman Plot Mean 95% CI Average 2.206 1.595-2.816 Washington1–Washington2 0.152 0.055-0.249 # of out of range (2SD): 3

128

Difference between McDade and DBS Washington1 Average = (McDade + DBS Washington1)/2 Difference = DBS Washington1 – McDade

Difference

-15

-12

-9

-6

-3

0

3

6

9

Average0 1 2 3 4 5 6 7 8 9 10 11 12

CRP

Bland-Altman Plot Mean 95% CI Average 1.808 1.128-2.487 Washington1–McDade 0.891 0.018-1.764 # of out of range (2SD): 2

129

Difference between McDade and DBS Washington2 Average = (McDade + DBS Washington2)/2 Difference = DBS Washington2 – McDade

Difference

-20

-10

0

10

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30

40

50

60

70

80

Average0 5 10 15 20 25 30 35 40 45 50

CRP

Bland-Altman Plot

Mean 95% CI Average 3.331 1.130~5.531 Washington2–McDade 2.742 -0.229~5.713 # of out of range (2SD): 2

130

Bland Altman Plots with Logged CRP Difference between Logged U Vermont Serum CRP and Logged Heritage DBP Average = (Logged Heritage + Logged Vermont Serum)/2 Difference = Logged Heritage – Logged Vermont Serum

Difference

-2.0-1.8-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0

Average-0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 2.1 2.3 2.5 2.7 2.9 3.1 3.3

CRP (with Vermont Serum and Heritage DBS)

Mean 95% CI Average 1.14 0.85 – 1.43 Heritage – Serum -0.84 -0.97 - -0.71 # of out of range (2SD): 1

131

Difference between Logged U Vermont Serum CRP and Logged Biosafe DBP Average = (Logged Biosafe + logged Vermont Serum)/2 Difference = Logged uvcrpdb2 (Biosafe) – logged uvcrpsrm

Difference

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

Average-0.7 -0.2 0.3 0.8 1.3 1.8 2.3 2.8 3.3 3.8

CRP (with logged Vermont Serum and logged Biosafe DBS)

Mean 95% CI Average 0.972 0.68 – 1.26 Biosafe – Serum -0.292 -0.36 - -0.22 # of Out of range (2SD): 2

132

Difference between Logged U Vermont Serum CRP and Logged McDade blood spot Average = (Logged Vermont Serum + logged McDade blood spot)/2 Difference = Logged McDade blood spot – Logged Vermont Serum

Difference

-2.0-1.8-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0

Average-0.3 0.2 0.7 1.2 1.7 2.2 2.7 3.2 3.7

CRP (with logged Vermont Serum and logged MCCRP)

Mean 95% CI Average -0.074 -0.41 – 0.27 McDade – Serum -0.494 -0.6 ~ -0.38 # of out of range (2SD): 1

133

Difference between logged Biosafe CRP and logged Heritage CRP Average = (Logged Biosafe + Logged Heritage)/2 Difference = Logged Heritage – Logged Biosafe

Difference

-2.0-1.8-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0

Average-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

CRP (with Logged Biosafe and Logged Heritage)

Mean 95% CI Average 0.938 0.65-1.23 Heritage – Biosafe -0.489 -0.61 ~ -0.37 # of out of range (2SD): 1

134

Difference between logged McDade and logged Heritage CRP Average = (Logged McDade + Logged Heritage)/2 Difference = Logged Heritage – Logged McDade

Difference

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

Average-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0

CRP (with Logged McDade and Logged Heritage)

Mean 95% CI Average 1.007 0.63-1.39 McDade – Heritage 0.370 0.13-0.61 # of out of range (2SD): 0

135

Difference between Logged McDade and Logged Biosafe CRP Average = (Logged McDade + Logged Biosafe)/2 Difference = Logged Biosafe – Logged McDade

Difference

-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0

Average-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

CRP (with Logged McDade and Logged Biosafe)

Mean 95% CI Average 0.802 0.42-1.18 McDade – Biosafe -0.147 -0.29 ~ -0.01 # of out of range (2SD): 1

136

Difference between Logged U Vermont Serum CRP and Logged DBS Washington1 Average = (Logged Washington1 + Logged Vermont Serum)/2 Difference = Logged Washington1 – Logged Vermont Serum

Difference

-3.0-2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.53.03.54.0

Average-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

CRP (with logged Vermont Serum and logged DBS_CRP1)

Bland-Altman Plot

Mean 95% CI Average 0.253 -0.022~0.529 Washington1 – Serum 0.407 0.292-0.522 # of out of range (2SD): 2

137

Difference between Logged U Vermont Serum CRP and Logged DBS Washington2 Average = (Logged Washington2 + Logged Vermont Serum)/2 Difference = Logged Washington2 – Logged Vermont Serum

Difference

-3

-2

-1

0

1

2

3

4

5

6

Average-3 -2 -1 0 1 2 3 4 5 6

CRP (with logged Vermont Serum and logged DBS_CRP2)

Mean 95% CI Average 0.397 0.080-0.714 Washington2 – Serum 0.423 0.276-0.570 # of Out of range (2SD): 2

138

Difference between logged Washington2 and logged Washington1 CRP Average = (Logged Washington2 + Logged Washington1)/2 Difference = Logged Washington1 – Logged Washington2

Difference

-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0

Average-4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

CRP (with logged DBS_CRP1 and logged DBS_CRP2)

Bland-Altman Plot

Mean 95% CI Average 0.342 0.062-0.623 Washington1 – Washington2 0.027 0.004-0.049 # of out of range (2SD): 2

139

Difference between logged McDade and logged DBS Washington1 CRP Average = (Logged McDade + Logged Washington1)/2 Difference = Logged Washington1 – Logged McDade

Difference

-3.0-2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.53.0

Average-4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

Mean 95% CI Average -0.157 -0.513~0.200 McDade – Washington1 -0.896 -1.072~-0.720 # of out of range (2SD): 2

140

Difference between Logged McDade and Logged DBS Washington2 CRP Average = (Logged McDade + Logged Washington2)/2 Difference = Logged Washington2 – Logged McDade

Difference

-4

-3

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-1

0

1

2

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Average-4 -3 -2 -1 0 1 2 3 4

CRP (with logged DBS_CRP2 and logged McDade)

Mean 95% CI Average 0.019 -0.375~0.412 McDade – Washington2 -0.915 -1.107~-0.723 # of out of range (2SD): 2

141

Figure 3. Differences in CRP by Serum level of CRP (without 1 outlier) (a)

CRP Difference

-14-12-10-8-6-4-2024

0 5 10 15 20 25

Vermont

Diff

Heritage - VTSerumBiosafe - VT Serum

(b)

CRP Difference

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Vermont

CRP

Diff

Vermont-HRSDBSVermont-BiosafeVermont-McDade

(c)

142

Comparison of Quartiles for Heritage DBS and VT Serum - No imputations for below detectability (N=30) Serum heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 23.33 (N=7)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

16.67 (N=5)

13.33 (N=4)

0.00 (N=0)

3rd qt 3.33 (N=1)

6.67 (N=2)

10.00 (N=3)

3.33 (N=1)

4th qt 0.0 (N=0)

0.0 (N=0)

3.33 (N=1)

20.00 (N=6)

70% in same quartile Quartiles 1st 2nd 3rd Heritage ~1.176 ~1.722 ~3.486 Serum ~2.930 ~3.905 ~7.820 Comparison of Quartiles for Biosafe DBS and VT Serum – No imputations for below detectability (N=41) Serum Biosafe

1st qt 2nd qt 3rd qt 4th qt

1st qt 19.51 (N=8)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt 7.32 (N=3)

21.95 (N=9)

2.44 (N=1)

0.00 (N=0)

3rd qt 0.00 (N=0)

2.44 (N=1)

21.95 (N=9)

0.00 (N=0)

4th qt 0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

24.39 (N=10)

88% in same quartile Quartiles 1st 2nd 3rd Biosafe ~0.966 ~2.184 ~3.192 Serum ~1.42 ~2.98 ~4.61

143

Comparison of Quartiles for McDade Blood Spot and VT Serum - No imputations for below detectability (N=60) Serum McDade

1st qt 2nd qt 3rd qt 4th qt

1st qt 20.00 (N=12)

5.00 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 5.00 (N=3)

15.00 (N=9)

5.00 (N=3)

0.00 (N=0)

3rd qt 0.00 (N=0)

5.00 (N=3)

16.67 (N=10)

3.33 (N=2)

4th qt 0.00 (N=0)

0.00 (N=0)

3.33 (N=2)

21.67 (N=13)

73% in same quartile Quartiles 1st 2nd 3rd McDade ~0.251 ~0.652 ~1.627 Serum ~0.420 ~0.970 ~2.965 Comparison of Quartiles for Heritage DBS and Biosafe DBS - No imputations for below detectability (N=29) Biosafe Heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 20.69 (N=6)

3.45 (N=1)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

13.79 (N=4)

17.24 (N=5)

0.00 (N=0)

3rd qt 6.90 (N=2)

3.45 (N=1)

10.34 (N=3)

0.00 (N=0)

4th qt 0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

24.14 (N=7)

69% in same quartile Quartiles 1st 2nd 3rd Heritage ~1.176 ~1.722 ~2.898 Biosafe ~1.974 ~2.856 ~4.788

144

Comparison of Quartiles for Heritage DBS and McDade Blood Spot - No imputations for below detectability (N=20) McDade Heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 10.00 (N=2)

15.00 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 5.00 (N=1)

10.00 (N=2)

10.00 (N=2)

0.00 (N=0)

3rd qt 10.00 (N=2)

0.00 (N=0)

10.00 (N=2)

5.00 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

5.00 (N=1)

20.00 (N=4)

50% in same quartile Quartiles 1st 2nd 3rd Heritage ~1.239 ~1.869 ~3.801 McDade ~1.510 ~2.754 ~6.474 Comparison of Quartiles for Biosafe DBS and McDade Blood Spot - No imputations for below detectability (N=27) McDade Biosafe

1st qt 2nd qt 3rd qt 4th qt

1st qt 22.22 (N=6)

3.70 (N=1)

0.00 (N=0)

0.00 (N=0)

2nd qt 3.70 (N=1)

14.81 (N=4)

7.41 (N=2)

0.00 (N=0)

3rd qt 0.00 (N=0)

7.41 (N=2)

7.41 (N=2)

11.11 (N=3)

4th qt 0.00 (N=0)

0.00 (N=0)

11.11 (N=3)

11.11 (N=3)

56% in same quartile Quartiles 1st 2nd 3rd Biosafe ~1.008 ~2.184 ~4.914 McDade ~0.988 ~1.710 ~4.130

145

Comparison of Quartiles for Logged values of Heritage DBS and VT Serum - No imputations for below detectability (N=30) Serum heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 20.00 (N=6)

6.67 (N=2)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

10.00 (N=3)

10.00 (N=3)

0.00 (N=0)

3rd qt 3.33 (N=1)

10.00 (N=3)

13.33 (N=4)

3.33 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

3.33 (N=1)

20.00 (N=6)

63% in same quartile Cutpoints for Quartiles 1st 2nd 3rd

Logged Heritage

~0.162 ~0.543 ~1.249

Logged Serum ~1.075 ~1.362 ~2.057 Comparison of Quartiles for Logged values of Biosafe DBS and VT Serum – No imputations for below detectability (N=41) Serum biosafe

1st qt 2nd qt 3rd qt 4th qt

1st qt 17.07 (N=7)

2.44 (N=1)

0.00 (N=0)

0.00 (N=0)

2nd qt 4.88 (N=2)

21.95 (N=9)

4.88 (N=2)

0.00 (N=0)

3rd qt 0.00 (N=0)

2.44 (N=1)

19.51 (N=8)

2.44 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

24.39 (N=10)

83% in same quartile Cutpoints for Quartiles 1st 2nd 3rd Logged biosafe ~ -0.035 ~0.781 ~1.161 Logged Serum ~ 0.351 ~1.092 ~1.528

146

Comparison of Quartiles for Logged values of McDade Blood Spot and VT Serum - No imputations for below detectability (N=60) Serum McDade

1st qt 2nd qt 3rd qt 4th qt

1st qt 20.00 (N=12)

5.00 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 5.00 (N=3)

15.00 (N=9)

5.00 (N=3)

0.00 (N=0)

3rd qt 0.00 (N=0)

5.00 (N=3)

16.67 (N=10)

3.33 (N=2)

4th qt 0.00 (N=0)

0.00 (N=0)

3.33 (N=2)

21.67 (N=13)

73% in same quartile Cutpoints for Quartiles 1st 2nd 3rd Logged McDade

~ -1.384 ~ -0.428 ~0.485

Logged Serum ~ -0.868 ~ -0.031 ~1.087 Comparison of Quartiles for Logged values of Heritage DBS and Biosafe DBS - No imputations for below detectability (N=29) Biosafe Heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 17.24 (N=5)

10.34 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

13.79 (N=4)

6.90 (N=2)

0.00 (N=0)

3rd qt 6.90 (N=2)

3.45 (N=1)

17.24 (N=5)

0.00 (N=0)

4th qt 0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

24.14 (N=7)

72% in same quartile Cutpoints for Quartiles 1st 2nd 3rd Logged Heritage

~0.162 ~0.543 ~1.064

Logged Biosafe ~0.680 ~1.049 ~1.566

147

Comparison of Quartiles for Logged values of Heritage DBS and McDade Blood Spot - No imputations for below detectability (N=20) McDade Heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 10.00 (N=2)

15.00 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 5.00 (N=1)

10.00 (N=2)

10.00 (N=2)

0.00 (N=0)

3rd qt 10.00 (N=2)

0.00 (N=0)

10.00 (N=2)

5.00 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

5.00 (N=1)

20.00 (N=4)

56% in same quartile Cutpoints for Quartiles 1st 2nd 3rd Logged Heritage

~0.213 ~0.625 ~1.335

Logged McDade

~0.412 ~1.003 ~1.864

Comparison of Quartiles for Logged values of Biosafe DBS and McDade Blood Spot - No imputations for below detectability (N=27) McDade Biosafe

1st qt 2nd qt 3rd qt 4th qt

1st qt 18.52 (N=5)

3.70 (N=1)

0.00 (N=0)

0.00 (N=0)

2nd qt 7.41 (N=2)

11.11 (N=3)

11.11 (N=3)

0.00 (N=0)

3rd qt 0.00 (N=0)

7.41 (N=2)

7.41 (N=2)

11.11 (N=3)

4th qt 0.00 (N=0)

0.00 (N=0)

11.11 (N=3)

11.11 (N=3)

48%in same quartile Cutpoints for Quartiles 1st 2nd 3rd Logged Biosafe ~0.008 ~0.781 ~1.592 Logged McDade

~ -0.012 ~ 0.536 ~1.418

148

Comparison of Quartiles for DBS Washington 1 and VT Serum - No imputations for below detectability (N=49) Serum Washington1

1st qt 2nd qt 3rd qt 4th qt

1st qt 26.53 (N=13)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

20.41 (N=10)

4.08 (N=2)

0.00 (N=0)

3rd qt 0.00 (N=0)

4.08 (N=2)

18.37 (N=9)

2.04 (N=1)

4th qt 0.0 (N=0)

0.0 (N=0)

2.04 (N=1)

22.45 (N=11)

88% in same quartile Quartiles 1st 2nd 3rd Washington 1 ~0.705 ~1.581 ~3.277 Serum ~0.42 ~1.01 ~2.54 Comparison of Quartiles for DBS Washington2 and VT Serum – No imputations for below detectability (N=52) Serum Washington2

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.00 (N=13)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

21.15 (N=11)

3.85 (N=2)

0.00 (N=0)

3rd qt 0.00 (N=0)

3.85 (N=2)

17.31 (N=9)

3.85 (N=2)

4th qt 0.00 (N=0)

0.00 (N=0)

385 (N=2)

21.15 (N=11)

85% in same quartile Quartiles 1st 2nd 3rd Washington2 ~0.729 ~1.706 ~3.678 Serum ~0.435 ~1.035 ~2.910

149

Comparison of Quartiles for DBS Washington1 and DBS Washington2- No imputations for below detectability (N=52) Washington2 Washington1

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.00 (N=13)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

23.08 (N=12)

1.92 (N=1)

0.00 (N=0)

3rd qt 0.00 (N=0)

1.92 (N=1)

23.08 (N=12)

0.00 (N=0)

4th qt 0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

25.00 (N=13)

96% in same quartile Quartiles 1st 2nd 3rd Washington1 ~0.594 ~1.537 ~3.238 Washington2 ~0.601 ~1.514 ~3.065 Comparison of Quartiles for DBS Washington1 and McDade Blood Spot - No imputations for below detectability (N=44) McDade Washington1

1st qt 2nd qt 3rd qt 4th qt

1st qt 18.18 (N=8)

6.82 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 6.82 (N=3)

13.64 (N=6)

4.55 (N=2)

0.00 (N=0)

3rd qt 0.00 (N=0)

4.55 (N=2)

18.18 (N=8)

2.27 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

2.27 (N=1)

22.73 (N=10)

73% in same quartile Quartiles 1st 2nd 3rd Washington1 ~0.576 ~1.305 ~2.976 McDade ~0.239 ~0.520 ~1.224

150

Comparison of Quartiles for DBS Washington2 and McDade Blood Spot - No imputations for below detectability (N=47) McDade Washington2

1st qt 2nd qt 3rd qt 4th qt

1st qt 19.15 (N=9)

6.38 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 6.38 (N=3)

14.89 (N=7)

4.26 (N=2)

0.00 (N=0)

3rd qt 0.00 (N=0)

4.26 (N=2)

17.02 (N=8)

4.26 (N=2)

4th qt 0.00 (N=0)

0.00 (N=0)

4.26 (N=2)

19.15 (N=9)

70% in same quartile Quartiles 1st 2nd 3rd Washington2 ~0.583 ~1.464 ~3.732 McDade ~0.245 ~0.589 ~1.544

151

Comparison of Quartiles for Logged values of DBS Washington1 and VT Serum - No imputations for below detectability (N=49) Serum Washington1

1st qt 2nd qt 3rd qt 4th qt

1st qt 24.49 (N=12)

2.04 (N=1)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

20.41 (N=10)

2.04 (N=1)

0.00 (N=0)

3rd qt 0.00 (N=0)

4.08 (N=2)

18.37 (N=9)

2.04 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

2.04 (N=1)

24.49 (N=12)

Cutpoints for Quartiles 1st 2nd 3rd

Logged Washington1 -0.350 0.458 1.187 Logged Serum -0.868 0.010 0.932 Comparison of Quartiles for Logged values of DBS Washington2 and VT Serum – No imputations for below detectability (N=52) Serum Washington2

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.0 (N=13)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

21.15 (N=11)

3.85 (N=2)

0.00 (N=0)

3rd qt 0.00 (N=0)

3.85 (N=2)

17.31 (N=9)

3.85 (N=2)

4th qt 0.00 (N=0)

0.00 (N=0)

3.85 (N=2)

21.15 (N=11)

Cutpoints for Quartiles 1st 2nd 3rd Logged Washington2 -0.318 0.534 1.302 Logged Serum -0.833 0.034 1.068

152

Comparison of Quartiles for Logged values of DBS Washington1 and DBS Washington2 - No imputations for below detectability (N=52) Washington2 Washington1

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.00 (N=13)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt 0.00 (N=0)

23.08 (N=12)

1.92 (N=1)

0.00 (N=0)

3rd qt 0.00 (N=0)

1.92 (N=1)

23.08 (N=12)

0.00 (N=0)

4th qt 0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

25.00 (N=13)

Cutpoints for Quartiles 1st 2nd 3rd Logged Wasington1 -0.522 0.429 1.175 Logged Washington2 -0.509 0.414 1.117 Comparison of Quartiles for Logged values of DBS Washington1 and McDade Blood Spot - No imputations for below detectability (N=44) McDade Washington1

1st qt 2nd qt 3rd qt 4th qt

1st qt 18.18 (N=8)

6.82 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 6.82 (N=3)

13.64 (N=6)

4.5 (N=2)

0.00 (N=0)

3rd qt 0.00 (N=0)

4.55 (N=2)

18.18 (N=8)

2.27 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

2.27 (N=1)

22.73 (N=10)

Cutpoints for Quartiles 1st 2nd 3rd Logged Washington1 -0.552 0.266 1.088 Logged McDade -1.434 -0.654 0.202

153

Comparison of Quartiles for Logged values of DBS Wasington2 and McDade Blood Spot - No imputations for below detectability (N=47) McDade Washington2

1st qt 2nd qt 3rd qt 4th qt

1st qt 17.02 (N=8)

6.38 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 8.51 (N=4)

14.89 (N=7)

2.13 (N=1)

0.00 (N=0)

3rd qt 0.00 (N=0)

4.26 (N=2)

19.15 (N=9)

4.26 (N=2)

4th qt 0.00 (N=0)

0.00 (N=0)

2.13 (N=1)

21.28 (N=10)

Cutpoints for Quartiles 1st 2nd 3rd Logged Washington2 -0.540 0.381 1.317 Logged McDade -1.406 -0.529 0.434

154

IMPUTED CRP Assigned .428 for undetectable cases (-333 code) in Heritage and Biosafe DBS); Assigned .10 for undetectable cases (-333 code) in VT Serum Comparison of Quartiles for Heritage DBS and VT Serum - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage DBS; .10 for undetectable cases (-333 code) in VT Serum) (N=86) Serum heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.58 (N=22)

24.42 (N=21)

15.12 (N=13)

0.00 (N=0)

2nd qt

3rd qt 0.00 (N=0)

0.00 (N=0)

8.14 (N=7)

3.49 (N=3)

4th qt 0.00 (N=0)

0.00 (N=0)

2.33 (N=2)

20.93 (N=18)

Cutpoints for Quartiles 1st 2nd 3rd Logged Heritage

~0.428 ~0.428 ~1.302

Logged Serum ~0.450 ~1.015 ~2.950 Comparison of Quartiles for Biosafe DBS and VT Serum – Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Biosafe DBS; .10 for undetectable cases (-333 code) in VT Serum) (N=82) Serum Biosafe

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.61 (N=21)

20.73 (N=17)

3.66 (N=3)

0.00 (N=0)

2nd qt

3rd qt 0.00 (N=0)

3.66 (N=3)

20.73 (N=17)

1.22 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

1.22 (N=1)

23.17 (N=19)

Cutpoints for Quartiles 1st 2nd 3rd Biosafe ~0.428 ~0.529 ~2.184 Serum ~0.420 ~0.945 ~2.980

155

Comparison of Quartiles for McDade Blood Spot and VT Serum - Imputations for below detectability (Assigned .10 for undetectable cases (-333 code) in VT Serum) (N=63) Serum McDade

1st qt 2nd qt 3rd qt 4th qt

1st qt 20.63 (N=13)

4.76 (N=3)

0.00 (N=0)

0.00 (N=0)

2nd qt 6.35 (N=4)

12.70 (N=8)

6.35 (N=4)

0.00 (N=0)

3rd qt 0.00 (N=0)

6.35 (N=4)

15.87 (N=10)

3.17 (N=2)

4th qt 0.00 (N=0)

0.00 (N=0)

3.17 (N=2)

20.63 (N=13)

Cutpoints for Quartiles 1st 2nd 3rd McDade ~0.218 ~0.593 ~1.544 Serum ~0.390 ~0.880 ~2.950 Comparison of Quartiles for Heritage DBS and Biosafe DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage and Biosafe DBS) (N=78) Biosafe Heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 48.72 (N=38)

2.56 (N=2)

11.54 (N=9)

0.00 (N=0)

2nd qt

3rd qt 0.00 (N=0)

0.00 (N=0)

10.26 (N=8)

2.56 (N=2)

4th qt 0.00 (N=0)

0.00 (N=0)

2.56 (N=2)

21.79 (N=17)

Cutpoints for Quartiles 1st 2nd 3rd

Heritage ~0.428 ~0.428 ~1.302 Biosafe ~0.428 ~0.630 ~2.184

156

Comparison of Quartiles for Heritage DBS and McDade Blood Spot - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage DBS) (N=59) McDade Heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.42 (N=15)

25.42 (N=15)

15.25 (N=9)

0.00 (N=0)

2nd qt

3rd qt 0.00 (N=0)

0.00 (N=0)

5.08 (N=3)

6.78 (N=4)

4th qt 0.00 (N=0)

0.00 (N=0)

5.08 (N=3)

16.95 (N=10)

Cutpoints for Quartiles 1st 2nd 3rd Heritage ~0.428 ~0.428 ~1.302 McDade ~0.232 ~0.629 ~5.948 Comparison of Quartiles for Biosafe DBS and McDade Blood Spot - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage DBS) (N=57) McDade Biosafe

1st qt 2nd qt 3rd qt 4th qt

1st qt 26.32 (N=15)

21.05 (N=12)

5.26 (N=3)

0.00 (N=0)

2nd qt

3rd qt 0.00 (N=0)

3.51 (N=2)

15.69 (N=9)

3.51 (N=2)

4th qt 0.00 (N=0)

0.00 (N=0)

3.51 (N=2)

21.05 (N=12)

Cutpoints for Quartiles 1st 2nd 3rd Biosafe ~0.428 ~0.428 ~1.974 McDade ~0.218 ~0.589 ~1.544

157

Comparison of Quartiles for Washington1 and Heritage DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage DBS) (N=48) Heritage Wash1

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.00 (N=12)

0.00 (N=0)

0.00 (N=0)

2nd qt

25.00 (N=12)

0.00 (N=0)

0.00 (N=0)

3rd qt 18.75 (N=9)

4.17 (N=2)

2.08 (N=1)

4th qt 0.00 (N=0)

2.08 (N=1)

22.92 (N=11)

Cutpoints for Quartiles 1st 2nd 3rd Heritage ~0.428 ~0.428 ~0.945 Washington1 ~0.590 ~1.537 ~3.238 Comparison of Quartiles for Washington1 and Biosafe DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Biosafe DBS) (N=51) Biosafe Wash1

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.49 (N=13)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt

21.57 (N=11)

1.96 (N=1)

1.96 (N=1)

0.00 (N=0)

3rd qt 1.96 (N=1)

1.96 (N=1)

21.57 (N=11)

0.00 (N=0)

4th qt 0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

23.53 (N=12)

Cutpoints for Quartiles 1st 2nd 3rd Biosafe ~0.428 ~0.630 ~1.806 Washington1 ~0.590 ~1.581 ~3.277

158

Comparison of Quartiles for Washington2 and Heritage DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage DBS) (N=51) Heritage Wash2

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.49 (N=13)

0.00 (N=0)

0.00 (N=0)

2nd qt

27.45 (N=14)

0.00 (N=0)

0.00 (N=0)

3rd qt 11.76 (N=6)

7.84 (N=4)

3.92 (N=2)

4th qt 0.00 (N=0)

1.96 (N=1)

21.57 (N=11)

Cutpoints for Quartiles 1st 2nd 3rd Heritage ~0.428 ~0.428 ~1.134 Washington2 ~0.605 ~1.694 ~3.623 Comparison of Quartiles for Washington2 and Biosafe DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Biosafe DBS) (N=54) Biosafe Wash2

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.93 (N=14)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt

18.52 (N=10)

3.70 (N=2)

3.70 (N=2)

0.00 (N=0)

3rd qt 1.85 (N=1)

0.00 (N=0)

20.37 (N=11)

1.85 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

1.85 (N=1)

22.22 (N=12)

Cutpoints for Quartiles 1st 2nd 3rd Biosafe ~0.428 ~0.672 ~1.974 Washington2 ~0.605 ~1.694 ~3.623

159

Comparison of Quartiles for Logged values of Heritage DBS and VT Serum - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage DBS; .10 for undetectable cases (-333 code) in VT Serum) (N=86) Serum heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.58 (N=22)

24.42 (N=21)

15.12 (N=13)

0.00 (N=0)

2nd qt

3rd qt 0.00 (N=0)

0.00 (N=0)

8.14 (N=7)

3.49 (N=3)

4th qt 0.00 (N=0)

0.00 (N=0)

2.33 (N=2)

20.93 (N=18)

Cutpoints for Quartiles 1st 2nd 3rd Logged Heritage

~ -0.849 ~ -0.849 ~0.264

Logged Serum ~ -0.799 ~0.015 ~1.082 Comparison of Quartiles for Logged values of Biosafe DBS and VT Serum – Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Biosafe DBS; .10 for undetectable cases (-333 code) in VT Serum) (N=82) Serum biosafe

1st qt 2nd qt 3rd qt 4th qt

1st qt 24.39 (N=20)

21.95 (N=18)

3.66 (N=3)

0.00 (N=0)

2nd qt 3rd qt 0.00

(N=0) 3.66

(N=3) 19.51

(N=16) 2.44

(N=2) 4th qt 0.00

(N=0) 0.00

(N=0) 1.22

(N=1) 23.17

(N=19) Cutpoints for Quartiles 1st 2nd 3rd Logged biosafe ~ -0.849 ~ -0.656 ~0.781 Logged Serum ~ -0.868 ~ -0.059 ~1.092

160

Comparison of Quartiles for Logged values of McDade Blood Spot and VT Serum - Imputations for below detectability (Assigned .10 for undetectable cases (-333 code) in VT Serum) (N=63) Serum McDade

1st qt 2nd qt 3rd qt 4th qt

1st qt 19.05 (N=12)

6.35 (N=4)

0.00 (N=0)

0.00 (N=0)

2nd qt 4.76 (N=3)

14.29 (N=9)

4.76 (N=3)

0.00 (N=0)

3rd qt 0.00 (N=0)

4.76 (N=3)

10.05 (N=12)

1.59 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

3.17 (N=2)

22.22 (N=14)

Cutpoints for Quartiles 1st 2nd 3rd Logged McDade

~ -1.523 ~ -0.523 ~ 0.434

Logged Serum ~ -0.942 ~ -0.128 ~ 1.082 Comparison of Quartiles for Logged values of Heritage DBS and Biosafe DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage and Biosafe DBS) (N=78) Biosafe Heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt

48.72 (N=38)

14.10 (N=11)

0.00 (N=0)

2nd qt

3rd qt

0.00 (N=0)

10.26 (N=8)

2.56 (N=2)

4th qt

0.00 (N=0)

2.56 (N=2)

21.79 (N=17)

Cutpoints for Quartiles 1st 2nd 3rd Logged Heritage

~ -0.849 ~ -0.849 ~0.264

Logged Biosafe ~ -0.849 ~ -0.462 ~0.781

161

Comparison of Quartiles for Logged values of Heritage DBS and McDade Blood Spot - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage DBS) (N=59) McDade Heritage

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.42 (N=15)

25.42 (N=15)

15.25 (N=9)

0.00 (N=0)

2nd qt

3rd qt 0.00 (N=0)

0.00 (N=0)

3.39 (N=2)

8.47 (N=5)

4th qt 0.00 (N=0)

0.00 (N=0)

5.08 (N=3)

16.95 (N=10)

Cutpoints for Quartiles 1st 2nd 3rd Logged Heritage

~ - 0.849 ~ -0.849 ~0.264

Logged McDade

~ -1.464 ~ -0.464 ~0.434

Comparison of Quartiles for Logged values of Biosafe DBS and McDade Blood Spot - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Biosafe DBS) (N=57) McDade Biosafe

1st qt 2nd qt 3rd qt 4th qt

1st qt 26.32 (N=15)

21.05 (N=12)

5.26 (N=3)

0.00 (N=0)

2nd qt 3rd qt 0.00

(N=0) 3.51

(N=2) 15.79 (N=9)

1.75 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

1.75 (N=1)

24.56 (N=14)

Cutpoints for Quartiles 1st 2nd 3rd Logged Biosafe ~ - 0.849 ~ - 0.849 ~0.680 Logged McDade

~ -1.523 ~ -0.529 ~0.434

162

Comparison of Quartiles for Logged values of Washington1 and Heritage DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage DBS) (N=48) Heritage Wash1

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.00 (N=12)

0.00 (N=0)

0.00 (N=0)

2nd qt

25.00 (N=12)

0.00 (N=0)

0.00 (N=0)

3rd qt 18.75 (N=9)

4.17 (N=2)

2.08 (N=1)

4th qt 0.00 (N=0)

2.08 (N=1)

22.92 (N=11)

Cutpoints for Quartiles 1st 2nd 3rd Logged Heritage ~ -0.849 ~ -0.849 ~ -0.057 Logged Washington1 ~ -0.528 ~0.429 ~1.175 Comparison of Quartiles for Logged values of Washington1 and Biosafe DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Biosafe DBS) (N=) Biosafe Wash1

1st qt 2nd qt 3rd qt 4th qt

1st qt 25.49 (N=13)

0.00 (N=0)

0.00 (N=0)

2nd qt

21.57 (N=11)

1.96 (N=1)

0.00 (N=0)

3rd qt 1.96 (N=1)

2.53 (N=12)

0.00 (N=0)

4th qt 0.00 (N=0)

1.96 (N=1)

23.53 (N=12)

Cutpoints for Quartiles 1st 2nd 3rd Logged Biosafe ~ -0.849 ~ -0.462 ~0.591 Logged Washington1 ~ -0.528 ~0.458 ~1.187

163

Comparison of Quartiles for Logged values of Washington2 and Heritage DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Heritage DBS) (N=51) Heritage Wash2

1st qt 2nd qt 3rd qt 4th qt

1st qt 23.53 (N=12)

0.00 (N=0)

0.00 (N=0)

2nd qt

25.49 (N=13)

0.00 (N=0)

0.00 (N=0)

3rd qt 15.69 (N=8)

5.88 (N=3)

3.92 (N=2)

4th qt 0.00 (N=0)

3.92 (N=2)

21.57 (N=11)

Cutpoints for Quartiles 1st 2nd 3rd Heritage ~ -0.849 ~0.849 ~0.126 Washington2 ~ -0.503 ~0.527 ~1.287 Comparison of Quartiles for Logged values of Washington2 and Biosafe DBS - Imputations for below detectability (Assigned .428 for undetectable cases (-333 code) in Biosafe DBS) (N=54) Biosafe Wash2

1st qt 2nd qt 3rd qt 4th qt

1st qt 24.07 (N=13)

0.00 (N=0)

0.00 (N=0)

0.00 (N=0)

2nd qt

20.37 (N=11)

3.70 (N=2)

0.00 (N=0)

0.00 (N=0)

3rd qt 1.85 (N=1)

0.00 (N=0)

22.22 (N=12)

1.85 (N=1)

4th qt 0.00 (N=0)

0.00 (N=0)

1.85 (N=1)

24.07 (N=13)

Cutpoints for Quartiles 1st 2nd 3rd Biosafe ~ -0.849 ~ -0.399 ~0.680 Washington2 ~ -0.503 ~0.527 ~1.287

164

1. Equations Linking Serum and DBS for CRP

CRP Equa y a b X R2 N VT Serum 0.01137 +2.296 Heritage 0.96 30 VT Serum 0.5529 +1.2146 Biosafe 0.98 41 VT Serum 0.3895 +1.1289 McDade 0.78 60 VT Serum 0.2074 +0.7260 Washington1 0.31 49 VT Serum 0.6816 +0.5188 Washington2 0.76 52 Comparison of Equations linking Serum and DBS when DBS assayed at Vermont have imputed values of .428 when below detection limit. CRP Equa y a b x R2 N VT Serum -0.20298 2.33508 Heritage 0.97 83 VT Serum 0.24341 1.23508 Biosafe 0.98 79

Conversions of DBS based on regression equations and Z scores When the values within detectible limits are estimated from the equation for 82 cases (for DBS Biosafe) and 86 cases (for DBS Heritage) including the imputed cases at .428 (.10 for serum), the means and standard deviation are shown below. Because everyone below .63 was coded at .428 which is estimated to be 0.74 (Biosafe) and 0.77 (Heritage), there are no values below this in the transformed sample. When DBS values are used with the regression equations to estimate the equivalent serum values, the means and standard deviations are very similar. The percent with high CRP (>=3.0) is quite close in the two samples.

(N=86) Mean SD Range Percent high

(>=3.0) Whole Blood 2.91 5.94 0.10-43.80 23.26 Serum est DBS Heritage 2.91 5.85 0.77- 42.89

23.26

(N=82) Mean SD Range Percent high

(>=3.0) Whole Blood 2.91 6.00 0.10-43.80 24.39 Serum est DBS Biosafe 2.91 5.93 0.74- 47.39

23.17

165

(N=49) Mean SD Range Percent high

(>=3.0) Whole Blood 1.57 1.63 0.16-8.03 14.29 Serum est DBS Washington1 1.57 1.59 0.22- 7.18

14.29

(N=52) Mean SD Range Percent high

(>=3.0) Whole Blood 3.06 6.89 0.16-43.80 19.23 Serum est DBS Washington2 3.06 6.81 0.56- 46.48

17.31

We can also make the conversion by changing the DBS values to Z scores and then convert the Z score into a serum blood value using the mean and SD of the distribution. The means and SDs are the same, and the percent with high CRP (>=3.0) is also similar for the transformed samples.

(N=86) Mean SD Range Percent high

(>=3.0) Whole Blood 2.91 5.94 0.10-43.80 23.26 Serum est DBS Heritage 2.91 5.94 0.74- 43.49

23.26

(N=82) Mean SD Range Percent high

(>=3.0) Whole Blood 2.91 6.00 0.10-43.80 24.39 Serum est DBS Biosafe 2.91 6.00 0.72- 47.91

23.17

(N=49) Mean SD Range Percent high

(>=3.0) Whole Blood 1.57 1.63 0.16-8.03 14.29 Serum est DBS Washington1 1.57 1.63 0.18-7.34

16.33

(N=52) Mean SD Range Percent high

(>=3.0) Whole Blood 3.06 6.89 0.16-43.80 19.23

166

Serum est DBS Washington2 3.06 6.89 0.53- 46.99

17.31

The percentage with high levels of CRP: With Biosafe Whole blood 24.39 DBS assay VT original 17.07 DBS used in regression est 23.17 DBS assay converted using Z scores 23.17 With Heritage Whole blood 23.26 DBS assay VT original 9.30 DBS used in regression est 23.26 DBS assay converted using Z scores 23.26

With Washington1 Whole blood 14.29 DBS assay VT original 28.57 DBS used in regression est 14.29 DBS assay converted using Z scores 16.33 With Washington2 Whole blood 19.23 DBS assay VT original 26.53 DBS used in regression est 17.31 DBS assay converted using Z scores 17.31

167

Figure 4. CRP in HRS 2006, 2008 and NHANES This section was done before the redone HRS values were available. HRS2008 has a number of people coded -999, -888, -444 codes and also -1. HRS 2006, over 1000 people have value of 0.03, this is the lower value of the assay. ug/ml is converted to mg/dl by dividing by 10.

The Figure below includes the distribution of CRP in NSHAP.

168

Because of the shape of the distribution of CRP, we also provide the distribution of the logged CRP value which is often used in analysis.