library(knitr)
library(tidyverse)

Introduction to Fill

Fill level is considered an important factor in coagulation tests because of the potential for the level of anticoagulant to influence the results of an assay. Coagulation test are particularly susceptible to influence from fill level because the tests are often positioned around the formation of a clot, as well as that the anticoagulant used has a much larger volume than other anticoagulants causing a potential for sample dilution. Although not all coagulation tests use clot formation as a means of measurement, some popular tests like heparin or d-dimer assays commonly use chromogenic or turbimetric methods.

3.2% sodium citrate is considered the standard anticoagulant for coagulation assays. The ratio of blood to anticoagulant is generally 9:1. It is an ideal anticoagulant for coagulation assays because its effects can be easily reversed to enable clotting to occur.

Fill level is accepted within certain margins based on the volume of anticoagulant in the tube. CSLI recommends a fill level within 10% of of the manufacturer recommended fill level (“CLSI Guideline, Dec. 2003, Doc. H1-A5, Vol. 23, No. 33.” 2003).

I’m starting off the blog with a series on the effect of fill volume on various types of coagulation testing. We will look into PT, aPTT, fibrinogen and common non clot based assays preformed on coagulation analyzers. In a review of the literature on the topic we will see how resilient tests are to under and over filled tubes, and what changes could be implemented in the clinical laboratory to accept more samples in critical situations without compromising the accuracy of the test.

Depending on the size of your facility a large amount of samples may potentially arrive with an improper fill level. Although a testing ban is commonly put in place for all improperly filled coagulation tube, rules are often ignored on the floor because of the anecdotal experience that tubes filled close to the acceptable level yield “close enough results” or fatigue from many improperly filled tubes. Those in reference labs may not even be aware of tube status from collection sites because they only work with aliquoted plasma samples.

Above are tubes with fill volumes of 3.5 mL, 3 mL, and 2 mL respectively.

fill_ratio_tbl <- tibble(
  "Percent_Filled" = seq(from = 50, by = 10, length.out = 11)
) %>% mutate("Ratio(Blood:Anticoagulant)" = (Percent_Filled/10)-1)
kable(fill_ratio_tbl)

Effects from Citrate Concentration

Lets first consider the effects of the varying concentrations of citrate found in tubes based on their fill level.

Citrate acts as an anticoagulant by the chelation of calcium from the plasma causing coagulation to be inhibited because the calcium dependent factors are unable to function. During a clot based assay additional calcium is added in the form of CaCl₂ to the plasma so that clotting can take place. An increased or decreased level of anticoagulant can change the amount of available calcium during the assay.

We can isolate the effect of citrate concentration of test results by looking at studies that compare 3.2% to 3.8% sodium citrate tubes. 3.8% citrate tubes are an alternative tube that is no longer used in standard practice.

Another source change of citrate concentration are from the varying hematocrits found in patient samples. Higher hematocrit samples have less plasma so they have more anticoagulant per unit of plasma. Free calcium is only found in the plasma portion of the sample, so patients with a higher hematocrit have less calcium to be chelated.

fill <- 50:120
hct <- 20:55

hct_fill_difference <- function (hct, fill) {
  citrate_vol <- 100/9

  plasma_percent <- (100-hct)/100
  plasma_vol <- plasma_percent*fill
  
  ac_ratio <- plasma_vol/citrate_vol
  
  return (ac_ratio)
}

ac_ratio <- outer(hct, fill, hct_fill_difference)

filled.contour(hct, fill, ac_ratio, plot.title=title(xlab="HCT", ylab="Fill %", main="Plasma:Citrate Ratio based on Fill Level and HCT"), nlevels=15)

Above I show the various levels of plasma to citrate solution ratio at different fill levels and hematocrit. As shown the ratio can vary greatly from around 4 to 8 in a acceptably filled specimen.

Dilutional Effects

Diltutional effects occur from the plasma being diluted by the anticoagulant when the fill volume deviates from expected. Dilutional effects on results are normally minor except in cases of extreme deviation from normal fill level.

What about hematocrit adjustment?

The practice of putting less citrate solution in tubes for patients is commonly preformed in laboratories when the patients hematocrit exceeds 55%. The purpose is to keep the ratio of plasma to citrate solution from varying too much by reducing the citrate volume. Adjustment techniques normally aim for a plasma to citrate ratio of 5.41(“CLSI Guideline, Jan. 2008, Doc. H21-A5, Vol. 28, No. 5.” 2008). Previously hematocrit adjustment was recommended by CLSI for for patients with hematocrits below 25% although this recommendation was removed due to lack of evidence.

Different tests have different effects from hematocrit adjustment, that have been looked at in various studies.

Tests

In future posts we will consider the effects of fill level on some of the most common coagulation test. We cannot isolate each of the many factors that can possibly influence the results of each test, but we can see the trends found in research that gathered the data to see the real world effects.

Bibliography

“CLSI Guideline, Dec. 2003, Doc. H1-A5, Vol. 23, No. 33.” 2003, December. https://fartest.ir/files/CLSI-H1-A5%20Tubes%20and%20Additives%20for%20Venous%20Blood.pdf#page=1&zoom=auto,-270,524.

“CLSI Guideline, Jan. 2008, Doc. H21-A5, Vol. 28, No. 5.” 2008, January.

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