Is it Christmas? Hematology Case Study: Coagulopathy

A 2 year old male was brought into the pediatrician’s office by his mother after tripping over a toy truck 2 days earlier. The mother stated that the child cut the inside of his lip in the fall, and the lip had been oozing blood for the past 2 days. The child had also experienced a bloody nose several times since the fall. Upon examination, the child appeared in general good health with no other bruising or bleeding. Examination of the joints revealed swelling in the right knee. The physician took a family history, and the mother reported that her younger brother has ‘some sort of bleeding problem’ and experienced prolonged bleeding after a tonsillectomy as a child, and after several surgeries as a young adult. The physician ordered blood work on the child.

  • Hgb 9.5 g/dl
  • Hct 30%
  • Platelet  185 x 103/ uL
  • INR  1.1
  • aPTT 57 sec
  • Mixing Test: corrected
  • Thrombin Time: normal

Based on these results, the prolonged aPTT warranted further investigation. A differential diagnosis involved ruling out other causes for the prolonged aPTT. The physician ordered mixing studies, factor VIII and factor IX assays and vWF. Mixing studies are used to determine if etiology of prolonged PT or PTT is due to a factor deficiency or an inhibitor. If the aPTT remains prolonged after mixing with normal plasma, this indicates an inhibitor. If the prolonged PTT becomes normal after the mixing studies, this would indicate a factor deficiency. The factor VIII and vWF were normal, but factor IX activity was 25%. Diagnosis: Factor IX deficiency. (It was also confirmed, after speaking with the child’s uncle, that he also had a factor IX deficiency)

So, you may ask, what does this have to do with Christmas? In the spirit of the season, I chose to present a Case Study on Factor IX deficiency, aka Christmas Disease. But, alas, this really has nothing to do with the holiday. Maybe it has something to do with the fact that the first article about this disorder was published in the British Medical Journal on Dec 27, 1954 (just 2 days after Christmas)? But, not so. Actually, Factor IX deficiency is also called Christmas Disease because it is named after Stephen Christmas, the first patient described to have Factor IX deficiency. Stephen Christmas was diagnosed with hemophilia in Toronto in 1949, at the age of 2. The family was visiting relatives in London in 1952 and it was there that doctors discovered that he was not deficient in Factor VIII, the cause of Classic Hemophilia as it was known at the time. It was discovered that he was deficient in another coagulation protein. This new protein was named Christmas protein and later became known as Factor IX.

A little bit more about the history of Factor IX deficiency. Before the discovery of the Christmas protein, it was thought that Hemophilia was a single disorder, caused by a deficiency of Factor VIII. With the discovery of this new protein, Classic Hemophilia (Factor VIII deficiency), was given the name Hemophilia A, and this new Factor IX deficiency became known as Hemophilia B. Yet another nickname for this disorder is the Royal Disease. Hemophilia was prominent in the European royal families in the 19rth and 20th centuries. Queen Victoria of Britain was a carrier of hemophilia and passed the gene on to three of her children. Her children and descendants married into the royal families of Germany, Russia and Spain, giving her the nickname the Grandmother of Europe. But, these marriages also served to spread the disease to these other royal houses, giving hemophilia the nickname Queen Victoria’s curse. The last known member of the royal families of Europe to carry the gene passed away in 1945, 9 years before that article in the British Medical Journal (December 27, 1954). So, how do we know that Hemophilia B is the hemophilia responsible for the Royal Disease? In 2009, DNA testing on bones identified as  Anastasia and Alexei Romanov, the last Russian royal family descendants of Queen Victoria, determined that the Royal Disease was Hemophilia B.

I remember teaching Hematology and Genetics before 2009 using a pedigree chart of Queen Victoria’s family to teach students about Hemophilia as an X linked recessive disorder. We created Punnett squares that showed the inheritance from Queen Victoria to her family members and descendants across Europe. I always enjoyed this lecture, because it was a fun piece of historical trivia paired with a good science lesson. After 2009, the science of the inheritance did not change, but we now knew that this Royal Disease was Hemophilia B. Hemophilia B is caused by mutations in the F9 gene which is responsible for making the factor IX protein.  The F9 gene is on the X chromosome. Hemophilia B, like Hemophilia A, is X linked, carried by the mother. 50% of males born to a carrier mother will have the disease and 50% of daughters will be carriers. All daughters of affected males will be carriers, but their sons will not be affected. Hemophilia A is more common than Hemophilia B, affecting about one in 5,000 males. Hemophilia B affects about one in 25,000 males. It has been though that up to about 30% of Hemophilia B cases occur as a spontaneous mutation and are not inherited. This has been thought to be the case with Queen Victoria. She has been believed to be ‘case zero’, the first hemophilia case in her family. However, some newer articles that have researched her family history suggest that she may have had a half-brother who had the disease.1 There are also other related disorders including a rare autoimmune acquired hemophilia B and another rare form of Hemophilia B called Hemophilia B Leyden.

The coagulation process involves many chemical reactions, from the initial event that triggers bleeding, to the formation of a clot. The sequence of events are generally depicted as a coagulation cascade to illustrate and simplify understanding of the process. The coagulation cascade is divided into 2 pathways, the intrinsic and extrinsic system, and a common pathway. This segregation of sections is not physiological, but allows for the grouping of factor defects and the interpretation of laboratory testing. Most problems with coagulation factors fall into one of three categories: a factor is not produced, there is a decreased production, or the factor is produced but not functioning properly. Hemophilia B is a factor IX deficiency. It is classified as mild, moderate or severe based upon the activity level of factor IX. In mild cases, bleeding symptoms may occur only after surgery or trauma and may not be diagnosed until later in life. In moderate and severe cases, bleeding symptoms may occur after a minor injury or even spontaneously. These moderate to severe cases are usually diagnosed at a younger age.

This child was diagnosed with Hemophilia B, based on coagulation studies, Factor IX assay results and family history. Treatment involves replacement of Factor IX to promote adequate blood clotting and prevent bleeding episodes.

References

  1. Turgeon, Mary Louise, Clinical Hematology: Theory & Procedures, 6th ed.  Lippincott Williams and Wilkins, Philadelphia, 2017.
  2. https://www.hog.org/publications/detail/the-royal-disease-a-family-history-update-on-queen-victoria
  3. https://rarediseases.org/rare-diseases/hemophilia-b/
  4. https://pediatriceducation.org/2015/12/14/what-is-it-called-christmas-disease/
  5. https://www.stago-us.com/hemostasis/tests-clinical-applications/hemophilia-b/

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Hematopathology Case Study: An 83 Year Old Man with an Elevated PTT

Case History

An 83 year old man with rapidly growing squamous cell carcinoma of the left temple and scalp underwent workup prior to surgery which showed an elevated PTT and a slightly elevated PT. The patient denied a history of abnormal coagulation tests or excessive bleeding or bruising. He also noted that he had previous surgeries including dental procedures without excessive bleeding. In addition, he did not have a history of clot formation.

Lab Values

Differential Diagnosis

At this point, the differential diagnosis for a prolonged PTT included the presence of an inhibitor (specific factor inhibiter vs. non-specific lupus anticoagulant) vs. reduced levels/activity of intrinsic pathway factors that would prolong the PTT, but would not significantly affect clot formation. This would include factors XI and XII. 

Additional Testing

An inhibitor screen/mixing study was performed and was positive. An inhibitor screen is performed by mixing the patient’s plasma with pooled normal plasma and running a PT or PTT.  If the PT/PTT corrects than the screen is negative. This means that a factor or factors were deficient in the patient’s plasma and were replaced with the pooled normal plasma resulting in a correction of the PT/PTT. In this case, a PTT at time 0 of 68 seconds and a PTT at 2 hours of 66 seconds was a failure to correct and indicated that an inhibitor was present, thus a positive result was entered.

The dilute Russell’s viper venom time (dRVVT) was used to test for a lupus anticoagulant. The screening test is performed by adding Russell viper venom, which directly activates coagulation factor X in the presence of calcium and a phospholipid poor reagent to the patient’s plasma and calculating time to clot. The confirmation test is the same assay with added excess phospholipid. In the presence of phospholipid dependent antibodies, the time to clot will be shorter for the confirmation test. The screen and confirmation ratios are normalized ratios (NR) of the patient sample result in seconds divided by the mean of the normal range in seconds. If the screen is <1.20, the confirmation test will not be run. If the screen is greater than 1.20 as seen here, the confirmation test will be run. The end result is reported as a normalized ratio of the screening test over the confirmation test. If the NR is greater than 1.20, than a lupus anticoagulant is reported as present.

Specific factor assays are performed by mixing the patient’s plasma with substrate plasma that is severely deficient in the factor being measured. Factor deficient plasma would be expected to give a prolonged clotting time. When patient plasma is mixed with factor deficient plasma, the clotting time will shorten and the degree of correction is proportional to the factor level in the patient’s plasma. The clotting times for the patient sample are compared to a reference curve. The reference curve is made with dilutions of normal plasma (containing 100% factor) added to factor deficient substrate plasma. All tests are run with 3 dilutions at 25%, 50% and 100% and curves are checked for parallelism errors, which might indicate the presence of an inhibitor. For this patient, factor XI was initially resulted as 1%, which would indicate a factor deficiency.

This is an example of a factor assay that shows parallelism. The reference plasma calibration curve and the patient plasma are parallel lines. 1

Analysis

From the results, it initially appeared that there was both a lupus anticoagulant and a factor XI deficiency. However, it would be odd for a patient with no reported coagulation abnormalities to suddenly have both a lupus anticoagulant and a factor XI deficiency. The raw data from the factor XI assay was obtained.

Upon review, the factor XI assay did show parallelism errors. Parallelism is tested by performing serial dilutions of a standard with known normal concentrations of factor and recording the time to clot. This line is shown with the red arrow. In contrast, the patient sample appears to be a flat line that is not parallel to the calibration curve. Parallelism errors were flagged because from the 50% to 25% dilution, the corrected results more than doubled. If there is a >20% change between dilutions, this indicates possible interference and additional dilutions should be run to dilute out the inhibitor. The 25% dilution had a corrected result of 2.9, which was greater than a 20% increase from the 50% dilution result of 1.3. Once more dilutions were performed; the Factor XI level was ultimately close to 100%.

Additional factors were checked to see if they also increased with dilutions. This would add support to the theory of a non-specific inhibitor (lupus anticoagulant) that was affecting all of the factor levels, rather than a specific factor XI inhibitor or a concurrent factor XI deficiency. The curve from factor IX (below) showed a similar phenomenon. As the sample underwent additional dilutions, the corrected result increased significantly (from 12.8 at 50% to 26.8 at 25%). Ultimately, the factor level was close to 82%.

The curve from factor VIII also showed low results to begin with and ultimately normal levels with additional dilutions. Altogether, this supported the presence of a strong lupus anticoagulant that was non-specifically interfering with all of the factor levels and prolonging the PTT.

Discussion

A prolonged PTT can be caused by many factors. In a patient without a bleeding history, lupus anticoagulant and certain factor deficiencies are high on the differential. The most common specific factor inhibitors are to FVIII and FIX. These generally arise in hemophilia patients treated with factor concentrates. It is very rare for a patient to develop an inhibitor to factor XI or XII.

Factor XI acts in the intrinsic pathway of the clotting cascade and is important for hemostasis. Deficiency of factor XI is rare and mainly occurs in Ashkenazi Jews. Generally, it does not cause spontaneous bleeding; however excessive blood loss can occur during surgical procedures.

Lupus anticoagulants are directed against proteins that complex with phospholipids. Although they prolong the PTT, they are associated with an increase in thrombosis rather than bleeding. In addition to interfering with the PTT assay, lupus anticoagulants may interfere with individual factor assays and result in non-parallelism (patient curve is not parallel to calibration curve) as seen in this patient. With increasing dilutions, the lupus activity will be disproportionately neutralized and the coagulation factor activity will increase in a non-parallel manner. 1

In a letter to the editor by Ruinemans-Koerts et al., they performed a set of experiments to investigate whether lupus anticoagulants vs. individual FVIII and FIX inhibitors can cause non-parallelism in the one-stage factor assay.  Non-parallelism was only detected using lupus sensitive reagents in plasma with high titers of lupus anticoagulants. The FVIII and FIX inhibitor containing samples both resulted in curves that were parallel to reference sample.

This curve shows that the factor IX inhibitor line is parallel to the reference plasma, while the lupus anticoagulant line is not. 1

Ultimately, this demonstrates the importance of running dilutions and being aware of parallelism errors when performing factor assays. This is especially important in patients with known or suspected lupus anticoagulants. In this case, the unlikely presence of a FXI deficiency with no previously reported coagulation testing abnormalities or bleeding history raised the suspicion of an inhibitor interfering with the factor assay. With a concurrent positive inhibitor screen and lupus anticoagulant test, as well as interference demonstrated with multiple factor assays, the best unified conclusion was a strong lupus anticoagulant. 1

References

  1. Ruinesman-Koerts, J., Peterse-Stienissen, I, and Verbruggen, B. ”Non-parallelism in the one-stage coagulation factor assay is a phenomenon of lupus anticoagulants and not of individual factor inhibitors. “ Letter. Thrombosis and Hemostasis, 2010, p.104.5.

Chelsea Marcus, MD is a Hematopathology Fellow at Beth Israel Deaconess Medical Center in Boston, MA. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

TEG and ROTEM

Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) are both laboratory methods for testing how well the blood coagulates. Rather than measuring the function or concentration of specific components of the coagulation pathways, like the PT or aPTT do, TEG and ROTEM measure the functional abilities of the overall coagulation pathways. They are intended to take into account everything that may be affecting coagulation, including such things as platelet function, fibrinolysis, illness, and medications. Thus both TEG and ROTEM are improvements on the old bleeding time test and also on the platelet function analysis (PFA).

In both cases reagents are added to a small sample of blood and the speed and size of clot formation is detected. Rotem works by inserting a pin into the sample and rotating the pin, with optical detection of the rotations. As clot formation occurs, it creates shear and drag on the pin, which is detected. TEG works essentially in the same way, but rotates the sample rather than the central pin. In both cases a set of parameters are measured and a visual graph of the coagulation is produced as the coagulation proceeds.

The main parameters that are produced and reported include:

  • R value – the reaction time in minutes, the time from beginning the test until clot formation begins
  • K value – the speed of clot formation in minutes, once clot formation has begun
  • Angle – a tangent drawn to the curve created as K is reached. This parameter is reported in degrees and gives mostly the same information as K. It is often reported rather than K.
  • Maximum Amplitude (MA) – the widest point of the curve in mm and an indication of the strength of the clot that has formed.
  • G value – the clot strength in dyn/cm2 .

These parameters and the shape of the graph that is produced can be interpreted in real time to indicate the efficiency of the patient’s coagulation system. TEG and ROTEM are most frequently used during surgery to keep track of the patient’s ability to coagulate and thus the level of anticoagulation being maintained. These are not tests that should be performed Point-of-Care even though surgeons who use this testing often like to see the shape of the curve as the clot formation occurs. Thus many places perform the test in the lab but have linked computer screens in the OR so that the surgeons can see the graph shape and parameter results in real time.

 

???????????????????????????????????????????????????????????????????????????????????

-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.

Heparin-Induced Thrombocytopenia

The fine folks over at The Poison Review discuss a paper about heparin-induced thrombocytopenia that is relevant to your interests. The paper appeared in the journal Clinical Toxicology and is geared toward that audience, but even so it serves as a nice refresher for technologists working in coagulation and hematology.

How Do We Monitor the New Anticoagulants-Podcast

As may or may not be aware, Lab Medicine has a podcast series geared toward laboratory professionals and pathologists. In a recent installment, Dr. Geoffrey Wool discusses the laboratory’s role in monitoring the new anticoagulants. Click this link to listen.