library(knitr)
library(tidyverse)
PT
I’m starting off the series by discussing the most common coagulation test the PT. Please read the intro post first to get an introduction to how we will be discussing fill volumes and ratios.
Based on research on the topic PT results are not significantly effect to a fill ratio of 7:1 in all patient populations. This goes beyond the CSLI and most manufacturer recommendations of having at least a 8:1 fill ratio. Considering most tubes that are underfilled are within this range, it presents an opportunity for reducing sample rejection rate substantially and improving patient care. This is especially relevant in high altitude settings where vacuum tubes frequently have insufficient pressure to be filled properly.
The PT is a clot based assay that uses thromboplastin to initiate clotting. The test can be used to identify various blood and liver disorders. In more critical situations the PT can be used to gauge the need for transfusion of FFP or PCC to a patient who is bleeding in cases of trauma or factor deficiencies (Khawar et al. 2018). Below we will look at studies that show how much the PT is effected by over and underfilling.
We will look at a series of papers that study the effect of fill level on PT results. We are only including data from 3.2% citrate tubes, as they are the modern standard, and 3.8% citrate tubes are known to be more sensitive to changes in fill volume. For each paper we will look at differences in patient results in terms of statistical significance as determined by the author, and clinical significance of greater than 15% in difference between a properly filled tube and underfilled tube as stated by Adock et al. [adcock1998minimum].
Underfilling
Healthy Patients
First we will look into the effects on PT results of healthy patients. Generally this means non hospitalized patients with a normal PT value who are not on anticoagulant therapy.
Adcock et al.
The paper by Adcock et al. analyzes changes with fill volume in both 3.2% and 3.8% citrate. We will use data from 3.2% citrate tubes. In the case of healthy patients the PT is not was significantly effected to a 60% fill volume or a 5:1 blood to anticoagulant ratio (Adcock, Kressin, and Marlar 1998).
Fill Limits:
Statistical Significance Limit: 5:1 Ratio
Clinical Significance Limit: 5:1 Ratio
Reneke et al.
A similar experiment was done by Reneke et al. where PT was studied using 3.2% citrate tubes. In the graph below the black circles represent healthy patients. The experiment showed similar results with little to no change in PT values of healthy patients down to a 60% fill volume or a 5:1 blood to anticoagulant ratio with both thromboplastin reagents that were tried. Data is harder to interpret from this study due to a lack of statistical analysis and tables (Reneke et al. 1998).
Fill Limits:
Unable to Determine
Odabasi et al.
Odabasi et al. showed no change in PT results to a fill level of 80% or a 7:1 ratio in healthy patients (Sena Odabasi and Yalcinkaya Kara 2023). I used the median data for this study because a P value was not provided in the mean data.
Fill Limits:
Statistical Significance Limit: 7:1 Ratio
Clinical Significance Limit: 6:1 Ratio
Lippi et al.
Lippi et al. paper performed data collection on 21 healthy patients and 6 oral anticoagulants. no significant or clinical change was found to the study limit for the PT [lippi2012quality].
Fill Limits:
Statistical Significance Limit: 5.7:1 Ratio (Study Limit)
Clinical Significance Limit: 5.7:1 Ratio (Study Limit)
Chuang et al.
Lastly, we will look at the study Impact of Evacuated Collection Tube Fill Volume and Mixing on Routine Coagulation Testing Using 2.5-mL (Pediatric) Tubes by Chuang et al. This study is our only one that utilized pediatric tubes. This study also showed no significant change in PT result to a 60% fill volume or 5:1 ratio. Although the authors attribute this lack of significance to a few healthy participants having elevated INRs. The authors also used a very low level alpha, so the results are likely significant at a level alpha of 0.05 (Chuang, Sadler, and Witt 2004).
The results being listed as INRs instead of the PT in seconds makes the comparisons difficult to perform with other studies, because we do not know the ISI or PT population mean that was used.
Fill Limits:
Statistical Significance Limit: 5:1 Ratio
Clinical Significance Limit: Unable to determine PT without ISI and Population Mean
Patients on Anticoagulant Therapy
In patients on vitamin K antagonist therapy effects from changes in fill volume are more pronounced but the research is not in complete agreement on the effects. Warfarin is used as a vitamin K antagonist. Vitamin K acts by adding carboxylic acid groups to glutamate present in coagulation factors (Imbrescia K 2023). The carboxylic acid groups allow the binding of calcium to coagulation factors. Warfarin therapy reduces vitamin K availability reducing the ability to bind calcium, and increasing sensitivity to the limited calcium found in under filled tubes.
The PT of patients on heparin are not effected in normal dosages because heparin primarily inhibits factor 8 invitro, but factor 8 does not play a major role in the PT. Most PT reagents also contain a heparin inhibitor to further reduce possible effects.
Adcock et al.
In the paper by Adcock et al. data on the PT of patients on unspecified oral anticoagulants (vitamin K antagonists given the publishing date) was collected in a second arm of the study [adcock1998minimum].
Fill Limits:
Statistical Significance Limit: Unspecified
Clinical Significance Limit: 7:1
Odabasi et al.
In the paper by Odabasi et al. patients on oral anticoagulants had no statistical difference, likely due to samaple size of 10. Although it appears a clinically significant change is present at a fill ratio below 7:1.
Fill Limits:
Statistical Significance Limit: 4:1 (Study Limit)
Clinical Significance Limit: 7:1
Overfilling
The effect of overfilling was not looked at in many papers. One paper using 3.8% citrate tubes by Pai et al. studied overfilling and found no difference up to a ratio of 12:1 [pai1990effect]. Extremely overfilled pediatric tubes likely do have an effect on results not seen in the above studies.
Analysis
Next I perform some analysis combining data from some of the above papers. From the papers discussed I was able to use data from Lippi et al., Adcock et al. and Odabasi et al. for healthy patient data. The paper by Lippi et al. does include some patients on anticoagulants, but the majority were healthy, so I used the data for the anticoagulant free population. For the patients on oral anticoagulants I used data from Adcock et al. and Odabasi et al.. None of the other papers were suitable for data usage.
A polynomial model was created with a degree of 2 for the patients on no anticoagulant and those on oral anticoagulants. The polynomial coefficients were both shown to be significant (p<0.05).
pt_df <- read_csv("pt_data.csv", show_col_types = FALSE)
ac_mod <- lm(CHANGE ~ poly(FILL_RATIO,2), data=filter(pt_df, AC_STATUS=="Oral Anticoagulants"))
summary(ac_mod)
##
## Call:
## lm(formula = CHANGE ~ poly(FILL_RATIO, 2), data = filter(pt_df,
## AC_STATUS == "Oral Anticoagulants"))
##
## Residuals:
## Min 1Q Median 3Q Max
## -0.16984 -0.04618 0.02839 0.06152 0.11922
##
## Coefficients:
## Estimate Std. Error t value Pr(>|t|)
## (Intercept) 0.37588 0.02862 13.135 1.24e-07 ***
## poly(FILL_RATIO, 2)1 -1.34721 0.10318 -13.057 1.32e-07 ***
## poly(FILL_RATIO, 2)2 0.64965 0.10318 6.296 8.95e-05 ***
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
##
## Residual standard error: 0.1032 on 10 degrees of freedom
## Multiple R-squared: 0.9546, Adjusted R-squared: 0.9455
## F-statistic: 105.1 on 2 and 10 DF, p-value: 1.935e-07
healthy_mod <- lm(CHANGE ~ poly(FILL_RATIO,2), data=filter(pt_df, AC_STATUS=="No Anticoagulants"))
summary(healthy_mod)
##
## Call:
## lm(formula = CHANGE ~ poly(FILL_RATIO, 2), data = filter(pt_df,
## AC_STATUS == "No Anticoagulants"))
##
## Residuals:
## Min 1Q Median 3Q Max
## -0.07028 -0.01429 -0.01029 0.03093 0.06282
##
## Coefficients:
## Estimate Std. Error t value Pr(>|t|)
## (Intercept) 0.071121 0.009931 7.161 4.85e-06 ***
## poly(FILL_RATIO, 2)1 -0.329657 0.040948 -8.051 1.27e-06 ***
## poly(FILL_RATIO, 2)2 0.160832 0.040948 3.928 0.00152 **
## ---
## Signif. codes: 0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1
##
## Residual standard error: 0.04095 on 14 degrees of freedom
## Multiple R-squared: 0.8514, Adjusted R-squared: 0.8302
## F-statistic: 40.12 on 2 and 14 DF, p-value: 1.597e-06
ggplot(mapping=aes(x=FILL_RATIO , y=CHANGE*100, col=AC_STATUS), data=pt_df) +
geom_point() +
geom_smooth(method = "lm", col="darkblue", formula = y ~ poly(x,2), data=filter(pt_df, AC_STATUS=="Oral Anticoagulants")) +
geom_smooth(method = "lm", col="darkred", formula = y ~ poly(x,2), data=filter(pt_df, AC_STATUS=="No Anticoagulants")) +
labs(
col = "Anticoagulant Status",
y = "Percent Change",
x = "Fill Ratio",
title = "Effect of Fill Ratio on PT in 3.2% Citrate Tubes"
)
Here we can see the two different relationships that exist depending on if the patient is on anticoagulants. Those on oral anticoagulants are more sensitive to underfilling. For both populations once the underfilling becomes extreme the quality of results rapidly deteriorates.
clin_lim <- tibble(FILL_RATIO=c(6.2))
predict(ac_mod, clin_lim)
## 1
## 0.1496109
clin_lim <- tibble(FILL_RATIO=c(4.6))
predict(healthy_mod, clin_lim)
## 1
## 0.147992
Ignoring the models confidence interval, the modeled fill limit for a clinically insignificant change (<15%) is a ratio of 6.2 for anticoagulated patients and 4.6 for healthy patients.
Discussion
Is it time to expand acceptance of tubes beyond the manufacturer recommendations?
The acceptance of specimens to a fill volume of 7:1 ratio is supported by the research for all patients regardless of medication status. Beyond 7:1 significant differences were shown by Odabasi et al..
Consideration must be taken to how this source of error could combine with instrument error and other collection errors that could influence a result to exceed the total allowable error. In the US CLIA allows a 15% error (Westgard, n.d.). In the results by Odabasi et al. percent difference was less than 10% in all patient populations when the tube was filled to a 7:1 ratio, and just below 15% when at 6:1 ratio.
Challenges exist in implementing a reduced fill level acceptance procedure due to the need for skilled workers to accurately compare the results against a visual guide. Validations must also be preformed to confirm the efficacy of procedures using that laboratories instrumentation.
In the emergency case it should be considered to expand the fill volume requirements. Most improperly filled tubes fall with the 7:1 acceptance policy, so it could make a substantial difference in rejection rates in emergency situations. Comments on results can be used to qualify the results, or special test codes can be implemented for emergency situations that loosen the fill level requirements.
Results are primarily effected in cases of high PT results. Selective release strategies can be implemented where results are only withheld if the PT exceeds a certain value, and normal PT results are released with a qualifying comment.
Bibliography
Adcock, Dorothy M, David C Kressin, and Richard A Marlar. 1998. “Minimum Specimen Volume Requirements for Routine Coagulation Testing: Dependence on Citrate Concentration.” American Journal of Clinical Pathology 109 (5): 595–99.
Chuang, Jung, Melanie A Sadler, and Daniel M Witt. 2004. “Impact of Evacuated Collection Tube Fill Volume and Mixing on Routine Coagulation Testing Using 2.5-Ml (Pediatric) Tubes.” Chest 126 (4): 1262–66.
Imbrescia K, Moszczynski Z. 2023. Vitamin k. [Updated 2023 Jul 10]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; https://www.ncbi.nlm.nih.gov/books/NBK551578/.
Khawar, Hina, Walt Kelley, Joshua B Stevens, and Nilmarie Guzman. 2018. “Fresh Frozen Plasma (FFP).”
Reneke, Johanna, Joan Etzell, Susan Leslie, Valerie L Ng, and Eugene L Gottfried. 1998. “Prolonged Prothrombin Time and Activated Partial Thromboplastin Time Due to Underfilled Specimen Tubes with 109 Mmol/l (3.2%) Citrate Anticoagulant.” American Journal of Clinical Pathology 109 (6): 754–57.
Sena Odabasi, Merve, and Zeynep Mine Yalcinkaya Kara. 2023. “Are Tube Fill Volumes Below 90% a Rejection Criterion for All Coagulation Tests?” Laboratory Medicine 55 (4): 442–46. https://doi.org/10.1093/labmed/lmad108.
Westgard, Sten. n.d. “Consolidated Comparison of Hematology and Coagulation Performance Specifications.” https://westgard.com/clia-a-quality/quality-requirements/hematology-goals.html.
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