Specimen Collection: HCT over 55%

Specimen Collection: HCT over 55%
George Fritsma
Jan 29, 2021 5:49pm

From Heidi Hobbs: Adjusting anticoagulant for hematocrits greater than 55% has been a hot topic lately. It is a challenge to ensure this is being done in all cases and we have some great things in the works.

While doing some research, I ran across one lab that has a simplified adjustment table, where patients with HCT 56–68% had one adjustment and patients with HCTs of 69% or more had another, but I can't verify how they determined their cutoffs. Also, a recent CAP education activity addressed this topic and had a statement pulled from the textbook Kitchen S, Olson JD, Preston FE. Quality in Laboratory Hemostasis and Thrombosis. 2nd ed . that says, "In samples with a HCT between 55% and 65%, a simplified method to overcome citrate effect is to remove 0.1 mL of the citrate anticoagulant from a standard 5 mL evacuated tube with a 4.5 mL blood draw volume and not perform the calculations. This will account for most of the patients with a high HCT , since very few patients will have a HCT that is greater than 65%. For those patients with HCT of greater than 65%, the above formula should be utilized to determine the correct anticoagulant volume."

This leads to further questions. Why is only removing 0.1 mL sufficient, when based on the calculation that isn't even quite sufficient for a HCT of 55%, let alone a HCT of 65%. If you were going to cover up to 65% you would need to remove 0.2 mL of anticoagulant from a 4.5 mL tube. Given the same logic as not making adjustments for low hematocrits, would you expect an adjustment for a HCT of 65% or even up to 70% have an insignificant effect on a HCT of 55%?

I appreciate any insight you have on this matter. Using tubes with a standard anticoagulant adjustment would be very helpful!! If we did go that direction, what sort of validation would need to be done, or is referencing the literature sufficient? Thanks!

If you pardon a self-reference, here is the relevant material from Fritsma GA, Laboratory evaulation of hemostasis. In Keohane EM, Otto CN, Walenga JM. Rodak's Hematology; Clinical Principles and Applications, Elsevier, 2020.

The 9:1 blood-to-anticoagulant ratio is effective, provided the patient’s hematocrit is 55% or below. In polycythemia the decrease in plasma volume relative to whole blood unacceptably raises the anticoagulant-to-plasma ratio, causing falsely prolonged clot-based coagulation test results. The phlebotomist must prepare specially marked tubes with relatively reduced anticoagulant volumes for collection of blood from a patient whose hematocrit is known to be 55% or higher. The anticoagulant volume may be computed by using this formula: C (1.85X10–3 ) (100–HCT ) V, where C is the volume of sodium citrate in mL, V is volume of whole blood-sodium citrate solution in mL, and HCT is the hematocrit in %.

For example, to collect 3 mL of blood/anticoagulant mixture from a patient who has a hematocrit of 65%, calculate the sodium citrate volume as follows: C (1.85X10–3) (100–65) 3.0mL = 0.19 mL of 3.2% sodium citrate. Remove the stopper from a 3-mL blue closure collection tube, pipette and discard 0.11 mL from the 0.3 mL of anticoagulant, leaving 0.19 mL. Collect blood in a syringe and transfer 2.81 (2.8) mL of blood to the tube, replace the stopper, and immediately mix by gently inverting at least three times.

Alternatively the phlebotomist can prepare for collection of 10 mL of blood and anticoagulant solution in a 12-mL centrifuge tube as follows: C (1.85X10–3 ) (100–65) 10.0 mL = 0.64 mL of sodium citrate. In this instance 0.64 mL of sodium citrate is pipetted into the tube, and 9.36 (9.4) mL of whole blood is transferred from the collection syringe.

This material is referenced to CLSI Standard H21-A5, H21; Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays, 5th Edition 2009.

I [Geo] admit that even when we were writing the Rodak instructions I had misgivings about how cumbersome the process must be. At the minimum, the method is likely to require a re-draw unless the phlebotomist somehow knew the patient's HCT in advance. Also, since most of us use 3 mL plastic vacuum tubes, how accurately can someone withdraw 0.11 mL of citrate? Or if we chose to prepare a 10 mL centrifuge tube, the phlebotomist would have to prepare a fresh tube for each patient collection. It seems like the CAP recommendation could solve our problems, provided the anticoagulant volume were validated against the CLSI standard's procedure.

So, as you can see, I don't have an answer, but have reached out to authors of Quality in Laboratory Hemostasis and Thrombosis. 2nd ed . and to Dennis Ernst, president of the Center for Phlebotomy Education and chair of the CLSI committee that prepared CLSI GP41: Collection of Diagnostic Venous Blood Specimens, 7th Edition. 2017. Meanwhile, I invite comments from our participants to learn how they've dealt with these specimens. Click on Comments below or email me at george@fritsmafactor.com.

Specimen Management
5 Comments

From Heidi Hobbs: Adjusting anticoagulant for hematocrits greater than 55% has been a hot topic lately. It is a challenge to ensure this is being done in all cases and we have some great things in the works.

While doing some research, I ran across one lab that has a simplified adjustment table, where patients with HCT 56–68% had one adjustment and patients with HCTs of 69% or more had another, but I can't verify how they determined their cutoffs. Also, a recent CAP education activity addressed this topic and had a statement pulled from the textbook Kitchen S, Olson JD, Preston FE. Quality in Laboratory Hemostasis and Thrombosis. 2nd ed . that says, "In samples with a HCT between 55% and 65%, a simplified method to overcome citrate effect is to remove 0.1 mL of the citrate anticoagulant from a standard 5 mL evacuated tube with a 4.5 mL blood draw volume and not perform the calculations. This will account for most of the patients with a high HCT , since very few patients will have a HCT that is greater than 65%. For those patients with HCT of greater than 65%, the above formula should be utilized to determine the correct anticoagulant volume."

This leads to further questions. Why is only removing 0.1 mL sufficient, when based on the calculation that isn't even quite sufficient for a HCT of 55%, let alone a HCT of 65%. If you were going to cover up to 65% you would need to remove 0.2 mL of anticoagulant from a 4.5 mL tube. Given the same logic as not making adjustments for low hematocrits, would you expect an adjustment for a HCT of 65% or even up to 70% have an insignificant effect on a HCT of 55%?

I appreciate any insight you have on this matter. Using tubes with a standard anticoagulant adjustment would be very helpful!! If we did go that direction, what sort of validation would need to be done, or is referencing the literature sufficient? Thanks!

If you pardon a self-reference, here is the relevant material from Fritsma GA, Laboratory evaulation of hemostasis. In Keohane EM, Otto CN, Walenga JM. Rodak's Hematology; Clinical Principles and Applications, Elsevier, 2020.

The 9:1 blood-to-anticoagulant ratio is effective, provided the patient’s hematocrit is 55% or below. In polycythemia the decrease in plasma volume relative to whole blood unacceptably raises the anticoagulant-to-plasma ratio, causing falsely prolonged clot-based coagulation test results. The phlebotomist must prepare specially marked tubes with relatively reduced anticoagulant volumes for collection of blood from a patient whose hematocrit is known to be 55% or higher. The anticoagulant volume may be computed by using this formula: C (1.85X10–3 ) (100–HCT ) V, where C is the volume of sodium citrate in mL, V is volume of whole blood-sodium citrate solution in mL, and HCT is the hematocrit in %.

For example, to collect 3 mL of blood/anticoagulant mixture from a patient who has a hematocrit of 65%, calculate the sodium citrate volume as follows: C (1.85X10–3) (100–65) 3.0mL = 0.19 mL of 3.2% sodium citrate. Remove the stopper from a 3-mL blue closure collection tube, pipette and discard 0.11 mL from the 0.3 mL of anticoagulant, leaving 0.19 mL. Collect blood in a syringe and transfer 2.81 (2.8) mL of blood to the tube, replace the stopper, and immediately mix by gently inverting at least three times.

Alternatively the phlebotomist can prepare for collection of 10 mL of blood and anticoagulant solution in a 12-mL centrifuge tube as follows: C (1.85X10–3 ) (100–65) 10.0 mL = 0.64 mL of sodium citrate. In this instance 0.64 mL of sodium citrate is pipetted into the tube, and 9.36 (9.4) mL of whole blood is transferred from the collection syringe.

This material is referenced to CLSI Standard H21-A5, H21; Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays, 5th Edition 2009.

I [Geo] admit that even when we were writing the Rodak instructions I had misgivings about how cumbersome the process must be. At the minimum, the method is likely to require a re-draw unless the phlebotomist somehow knew the patient's HCT in advance. Also, since most of us use 3 mL plastic vacuum tubes, how accurately can someone withdraw 0.11 mL of citrate? Or if we chose to prepare a 10 mL centrifuge tube, the phlebotomist would have to prepare a fresh tube for each patient collection. It seems like the CAP recommendation could solve our problems, provided the anticoagulant volume were validated against the CLSI standard's procedure.

So, as you can see, I don't have an answer, but have reached out to authors of Quality in Laboratory Hemostasis and Thrombosis. 2nd ed . and to Dennis Ernst, president of the Center for Phlebotomy Education and chair of the CLSI committee that prepared CLSI GP41: Collection of Diagnostic Venous Blood Specimens, 7th Edition. 2017. Meanwhile, I invite comments from our participants to learn how they've dealt with these specimens. Click on Comments below or email me at george@fritsmafactor.com.

By steve
Feb 1, 2021 5:30am
From Dr. Steve Kitchen, Clinical Scientist, Dept of Coagulation, Royal Hallamshire Hospital, Sheffield: ICSH are constructing some up to date guidance on this topic. below is close to the final wording that will be used. it is under review with IJLH just now.
Besides filling problems, an additional issue may compromise the balance between citrate and ionized calcium in the specimen. Hematocrit (HCT) is conventionally defined as the ratio between the volume of red blood cells (RBCs) and the total volume of blood. The greater is the HCT value, the lower is the amount of plasma and its constituents in the total volume of blood. When the HCT value exceeds 0.55 (55%), the concentration of ionized calcium in the blood tubes is so increased that clotting tests may be ultimately impaired with possible spurious prolongation of PT and APTT (68). Such samples have an altered anticoagulant to plasma ratio in a similar way to underfilled samples as you are anticoagulating the plasma component not the cellular component. This problem is avoided by altering the anticoagulant to blood ratio so that less anticoagulant is added. Clinical and Laboratory Standards Institute (CLSI) has released a specific recommendation (H21 A5) that a formula should be used for adjusting the final concentration of citrate in the blood tube whenever the HCT is greater than 55%:
residual volume of citrate in the tube = [100%–HCT] X sample volume/[595-HCT in % ] (63), a formula described in a study assessing the impact of citrate concentration on PT (69). Adjustment of citrate to blood ratio when HCT is greater than 55% is also recommended by the World Federation of Haemophilia for coagulation test related to investigation of subjects with bleeding disorders (70). An alternative formula has been described as follows (24):
C = (1.85 x 10-3)(100–HCT )(V), where C is the volume of citrate in millilitres (mL) that should be added to a volume of blood (V) in mL to form an anticoagulated blood sample. Using an example:
HCT of 70% and 4.5 mL of collected blood prior to addition of anticoagulant, this gives the following calculation: 1.85 x 0.001 x 30 (100–70) x 4.5 mL = 0.25 mL of citrate, thus 0.25 mL of citrate is mixed with 4.5 mL of blood.
This formula has been validated for use with PT, APTT and other coagulation tests (68), so should be preferred although the calculated citrate to blood ratio using the two formulas is practically identical.
If a patient is severely anemic, there is an increased plasma volume so that there may be sufficient residual calcium after mixing with trisodium citrate in the tube for coagulation to proceed in the sample, leading to activation and possible shortening of APTT alongside consumption of clotting factors, including fibrinogen. However it seems that low hematocrit has less effect on results so there is usually no requirement to adjust the citrate to blood ratio for samples from subjects with anaemia (71).
Recommendation 10.1: The ratio of blood to trisodium citrate anticoagulant should be adjusted for coagulation tests when patients have hematocrit >55% (>0.55) using the formula C = (1.85 x 10-3)(100-Hct )(V) where C is the volume of citrate in mL that should be added to a volume of blood (V) in mL to form an anticoagulated blood sample.
Remark: Once a tube has been specifically prepared for use in a particular patient it should be labelled and handled in such a manner that will be only used for the intended patient.
By Director Dennis Ernst
Feb 1, 2021 10:01am
Adjusting citrate concentrations has always been a conundrum and probably always will be. While I agree the calculation provided by Dr. Kitchen may be more precise, the practice of simply removing 0.1 mL for all patients with HCTs 55%-65% seems more practical. Yet I am of the opinion the established standards must prevail and the calculations performed. That said, it is my opinion the method of adjustment is not as problematic as the overarching problem: is this a preanalytic or analytic concern?

The answer is, of course, both. But it's no secret in our industry there's a wall in many facilities obstructing cooperation and communication between the testing and collection personnel. That's a conversation for another day and on another blog.

In labs with good intradepartmental cooperation, the preanalytic staff (phlebotomists) are not going to make the calculation nor the adjustment. It's more than likely the analytic staff (testing personnel) prepare the tube, hand it to the phlebotomist and says "here, use this." Then the phlebotomist incurs the risk of drawing into a syringe (a device more likely to inflict a needlestick), pop the stopper on the modified citrate tube and squirt it in to the fill mark (another opportunity for an exposure since we now have an open system of transfer). Until tube manufacturers create special coag tubes for patients with high hematocrits in which the vacuum is intact, the risk to the phlebotomist is greater than it should be. But the market for a "high-hematocrit citrate tube" is likely too small to make production appealing to tube makers or affordable to laboratories.

There's yet one more problem with getting properly citrated tubes into the hands of phlebotomists. In most labs, the one performing the coag testing likely has no idea which patients have high hematocrits unless there's a mechanism in place that communicates it to the coag-testing tech. If the hematology department isn't verbally notifying the coag personnel or the coag personnel isn't checking the LIS for high hematocrits on every batch or random assay, over-citrated coag results get reported on patients with high hematocrits. It is my opinion this communication breakdown runs rampant. If I'm wrong, I'd love to hear an opposing viewpoint.

Thanks for the opportunity to opine.
By MD John Olson
Feb 2, 2021 6:21pm
From Dr. John D. Olson: The reference to removing 0.1 mL (20%) of the anticoagulant from a 5 mL collection tube without releasing the vacuum is in the book chapter “Sample Integrity and Preanalytical Variables” from Kitchen S, Olson JD, Preston FE. Quality in Laboratory Hemostasis and Thrombosis. 2nd ed (p 49). The chapter was contributed by Dr. Adcock. This is a direct quote from the CLSI document H21-A5: Collection, Transport, and Processing of Blood Specimens for Testing Plasma-based Coagulation Assay and Molecular Hemostasis Assays. (pp 8-9). The source is not cited in the CLSI document.
Dr. Richard Marlar may be the source of the statement in the CLSI document. He has generated data for the method but has not published the information. He has allowed me to provide this comment from his email reply to a query from Dr. Adcock and me:
“I (Dr. Marlar) published a 2006 paper in AJCP about high HCT and volume adjustment. We said in that paper, to use a formula for removing citrate. In real practice we found the process to be inexact and difficult to extract the correct volume. So we decided to do it as a standard amount removed. We removed 20% of the anticoagulant volume and then we made the tubes up and stored them for when the need arose. I stated that in an article that Bob Gosselin and I (Dr. Marlar) published a review of pre-analytical variables for Seminars in Thrombosis and Hemostasis (2019). No data is presented in that article. Does not help now (as we have not published it) but we are writing a paper on the theoretical work comparing citrate concentration with high HCT and using the formula versus set amount of 20%. When I was in Oklahoma, we validated the removal of 20% of the citrate while keeping the vacuum in the tube versus the formula for removal. We found no difference butwe never published the data.” [From Richard Marlar, 2/2/21.]
When one looks at the range of plasma volumes collected in the same volume of citrate, it is easy to see that concentration of citrate is widely variable. This is particularly apparent with increasing [anemia]. This argues that we are likely using a higher concentration of citrate than necessary and that the adjustment of the concentration does not demand great precision. If you do the math, the removal of 20% of the volume of citrate would easily cover specimens between HCT 55% and 65%. Making this process easy will provide patient safety and will be much more likely to lead to clinical/laboratory compliance. Thanks for the opportunity to comment.
By Twyla
Feb 2, 2021 11:13pm
I'm no longer in the hematology department (moved to IT/Clinical Operations) but I set up the procedure for our high HCT coagulation specimens years ago when I was supervisor.
In our hospital system, we had a reasonably closed population of about 150,000 people. Every hospital and lab location within our health authority was on the same LIS/CIS, thankfully.
We had a rule set up on the CBC bench to alert to a HCT >55% (0.55 L/L in Canadian units) to remind the technologist to check for a corresponding coagulation specimen. If there was a coag specimen from the same requisition, it would prevent the autorelease of the coag results to give us time to add comments as needed. This required communication between routine hematology and coag.
We also had a system of markers that we could add to a patient's chart to quickly alert techs to the "known" high HCT patients and we gave the patient a card to carry with their healthcare insurance card.
The first time that we encountered a patient with a high HCT, we would add comments to the coag specimen (if there was one) advising a recollection in an adjusted citrate tube. Since our patients generally tend to attend the same collection location, we would prepare two or three adjusted tubes labeled with neon pink stickers to differentiate them from regular tubes and identify the patient they were meant for based on their most recent HCT result and send them to that site. We would also give the patient a card to bring with them identifying them as needing an adjusted coag tube collected.
When the patient arrived, we had the phlebotomist collect a regular citrate tube, the adjusted citrate tube, and a CBC so we could ensure that the proper adjustment was made based on the current actual HCT.
We would run both PT and PTT on the unadjusted tube and the adjusted tube in order to confirm that the results were falsely prolonged in the unadjusted tube. They always were, particularly the PTT. This was our internal QA check.
One trick that I came up with over the years was to use a tuberculin syringe/needle to withdraw the citrate from the adjusted tube through the lid of the tube. This meant that the blood could still be collected using a Vacutainer system (don't over-fill!) because the adjusted citrate tube still had a vacuum. As long as we could see that the PTT result corrected from prolonged in the unadjusted tube to within reference range in the adjusted tube, the INR was considered reportable. Most often outpatient coag is ordered for PT to monitor Coumadin, of course.
One of our more severe polycythemia patients commented to me one day that he far preferred to come to our lab than the lab in the much bigger city, as we only needed to poke him once and they always poked him twice.
The procedure/process required a few tweaks to our LIS in order to work, but it was better for patients as they weren't poked twice and we could prove that the adjustment to the citrate volume was adequate based on the HCT and the correction of the PTT.
By George Fritsma
Feb 5, 2021 12:50pm
From George, thanks to Dr. Kitchen, Dennis Ernst, Dr. Olson with reference to Dr. Marlar, and Twyla. Some years ago I spoke with a medical laboratory scientist who said their lab only reduces anticoagulant volume when the PT or PTT was prolonged. A result within the normal interval would be reported without reference to the HCT. I can find no fault with this logic, but would like our participants to describe a potential situation where this could result in an erroneous report.

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