Tag: coagulation

Coagulation Case Study: A 31 Year Old Female with Bleeding Gums, Bruising, and Recurrent Nosebleeds

Introduction

Disseminated intravascular coagulation (DIC) is a serious process in which the proteins responsible for clot formation become overactive. It is vital to note that DIC is not a disease in itself, rather it is always secondary to an underlying disorder, which causes over-activation of the coagulation system.1 This over-activation results in activation of coagulation that consumes platelets, clotting factors, and causes microvascular fibrin thrombi, which can lead to organ failure secondary to tissue ischemia.2 The brain, kidneys, liver, and lungs are the most prone to tissue damage in DIC.

Some symptoms of DIC may include bleeding (from multiple sites), blood clots, bruising, hypotension, shortness of breath, fever, confusion, memory loss, or behavior change.3 Patients always present with symptoms of other disorders frequently associated with DIC such as sepsis, trauma, liver disease, obstetric complications, and malignancy to name a few. In most cases, coagulation does not result in clinical complications, and is not evident until the consumption of platelets and factors becomes overwhelming.1 Once there is overwhelming consumption, it becomes visible through prolonged clotting tests and increasing thrombocytopenia.1 Therefore, timely recognition is crucial in recognizing DIC.

A major challenge of both prognosis and management, of DIC is that it is not a single disease entity. Prognosis largely depends on the treatment of the underlying condition, as the condition is what drives the activation of the coagulation cascade. Treatment is generally supportive, and entails supporting various organs. Support may include the use of ventilators, hemodynamic support, or transfusion of blood components. As well, algorithms have been developed to help guide the management of patients with DIC.

Case presentation

A 31-year-old female presented to the emergency department with complaints of bleeding gums, bruising all over her body, and recurrent nosebleeds for longer than a week. The patient has a history of Acute Promyelocytic Leukemia (APL/AML-M3), and has had symptoms of gingival bleeding, widespread ecchymosis, and recurrent epistaxis for longer than a week. Past fluorescence in situ hybridization (FISH) of the patient’s bone marrow aspirate revealed a t(15;17) translocation. Bone marrow studies also showed blast cells with granulation, dysmyelopoiesis, and 60% promyelocytes with the presence of Auer rods. Immunophenotyping was negative for expression of CD34, and positive for expression of CD13 and CD33. Cytochemical staining tests were positive with myeloperoxidase (MPO), Sudan Black B (SBB), and specific esterase (SE), and negative with non-specific esterase (NSE). As well, reverse transcriptase-polymerase chain reaction (PCR) confirmed the PML-RAR(alpha) fusion gene, consistent with a diagnosis of AML-M3.

The patient has undergone one past treatment of retinoic acid, as well as traditional chemotherapy. It should be noted that some studies indicate a higher incidence of DIC in acute leukemia patients during remission induction with chemotherapy.1 The patient has no family history of the disorder/condition. There are no occupational or social implications/factors, but it is associated with a specific genetic mutation. The patient has no known drug allergies.

Vital signs were normal upon arrival. Upon physical examination, the patient appeared pale and lethargic, displayed widespread ecchymosis, and swollen gums.

Laboratory tests revealed a decreased hemoglobin concentration of 6.0 g/dL (normal, 12.0-16.0 g/dL), and an increased white blood cell (WBC) count of 19.5 x 109/L (normal, 4.5-11.0 x 109/L). The WBC differential showed 91% promyelocytes with heavy granulation, 3% myelocytes, 4% metamyelocytes, and 2% neutrophils. Platelet count was decreased at 72,000/uL (normal, 150,000-450,000/uL). Prothrombin time (PT) was prolonged at 22 seconds (normal, 10-13 seconds) and activated partial thromboplastin time (aPTT) was prolonged at 54 seconds (normal, 23-36 seconds). D-dimer concentration showed an increase at 12 ug/mL (normal, <0.5 ug/mL), and a decreased fibrinogen concentration of 60 mg/dL (normal, 200-400 mg/dL). Plasma levels of plasminogen were 65% (normal, 80-120%), and plasma levels for a2-antiplasmin were 46% (normal, 80-100%). These findings are consistent with DIC with marked hyperfibrinolysis, as correlated by low levels of a2-antiplasmin and plasminogen. The patient was diagnosed as having symptoms associated with DIC secondary to AML-M3.

The treatment for this patient included avoidance of all invasive procedures including biopsies or intravenous (IV) line placement, as the patient was at high risk for bleeding. Likewise, due to the high risk of bleeding, the use of heparin as a supportive therapy was not advocated. The patient received a platelet transfusion, with an aim of a platelet count greater than 50 x 109/L. As well, the patient received fresh frozen plasma (FFP) and fibrinogen concentration, guided by the concentration in the patient’s plasma. Supportive treatment will continue to be maintained during remission induction of AML-M3, and the disappearance of the coagulopathy.

Discussion

The typical symptoms of DIC are generally those of the underlying process, which incites the condition. Underlying conditions may include, but are not limited to, severe infection with any microorganism, sepsis, trauma, solid tumors and malignancies, hepatic failure, and obstetric complications.4 Acute DIC may present as ecchymosis or petechiae. Additionally, there is usually a patient history of bleeding such as gastrointestinal or gingival bleeding.4 Post-operative DIC patients can also experience bleeding from various surgical sites, or within serous cavities.5 One should remain cognizant of signs and symptoms of deep vein thrombosis (DVT), as well as microvascular thrombosis. One hallmark of DIC is bleeding from at least three unrelated sites.4 Chronic DIC may present as thrombosis due to excess thrombin formation. Patients with comorbid liver disease may also present with jaundice, while patients with pulmonary involvement may present with a cough, dyspnea, and hemoptysis.4

Epidemiology

DIC occurs in all ages and races, and has not been shown to have a predisposition for a particular gender.4 DIC has been known to develop in about 1% of all hospitalized patients.4 DIC accounts for 9%-19% of intensive care unit (ICU) admissions.2 Although assigning values to DIC-related morbidity and mortality proves difficult, studies show it occurs in roughly 35% of septic patients.1 In a trauma setting, the presence of DIC may often double the mortality rate.4 DIC may be diagnosed in roughly 15% of patients with underlying acute leukemia.1 Previous studies report the occurrence of major bleeding to be 5%-12%, although patients with a platelet count of less than 50 x 109/L show a higher risk for bleeding when compared to patients with increased platelet counts.1 The occurrence of thrombosis in both small and midsize vessels is more commonly seen, which contributes to organ failure. Overall, the severity of DIC is directly related to increased mortality, thus emphasizing the importance of early recognition.

Related Disorders/Conditions

Several conditions are known to be complications of DIC. These conditions include, but are not limited to, kidney injury, altered mental status, respiratory dysfunction, hemothorax, gangrene, digit loss, and shock.4 In most cases, the release of tissue material into circulation in conjunction with endothelial disruption, leads to the activation of the coagulation cascade.

Pathophysiology

In DIC, the patient’s underlying condition stimulates powerful procoagulant activity that results in excess thrombin. The excess thrombin then overcomes the anticoagulant control mechanisms of protein C (PrC), antithrombin (AT), and tissue factor pathway inhibitor (TFPI), allowing for widespread thrombosis throughout the vasculature.2 The patient paradoxically battles between an excess thrombin state due to thrombin, embolism, and microvascular occlusion, and a hemorrhagic disorder secondary to depletion of platelets, consumption of coagulation factors, and accelerated formation of plasmin.2

To fully understand DIC, one must review normal hemostasis. Normal hemostasis generally takes place in response to an injury to the blood vessel wall. In normal hemostasis, a well-maintained process takes place in three stages: primary hemostasis, secondary hemostasis, and fibrinolysis. Primary hemostasis results in formation of the primary platelet plug via platelet adhesion, aggregation, and secretion.6 Secondary hemostasis is responsible for the formation of fibrin, and involves plasma proteins. In secondary hemostasis, fibrinogen is converted to fibrin, which forms a mesh that acts to reinforce the primary platelet plug, thus forming a hard, more stabilized clot at the site of injury, stopping any active bleeding.6 Finally, fibrinolysis takes place. Fibrinolysis is the process of the removal of fibrin through proteolysis, so that normal vessel structure and function can be restored.6 As part of normal healing, in response to an injury, clotting should only occur as a localized phenomenon, as there are many checks and balances to prevent extension of the hemostatic plug mechanism to the entire intravascular system.2 In DIC, this process of hemostasis runs out of control.

Laboratory Tests and Applications

Since DIC is secondary to an underlying disease, its diagnosis can be challenging, as there is no single laboratory test with sufficient accuracy to allow for a diagnosis. Imaging studies are generally only useful in detecting the underlying condition inciting DIC.4 DIC may present with a wide range of abnormalities in laboratory values. Laboratory findings of thrombocytopenia, prolonged coagulation times, decreased fibrinogen, elevated fibrin degradation products (FDPs), and elevated D-dimer levels are indicative of DIC.4 A complete blood count (CBC) and peripheral blood smear may show moderate-to-severe thrombocytopenia, as well as the presence of schistocytes secondary to evidence of microangiopathic pathology.4 Additionally, scoring systems have been developed to aid in diagnosis of DIC.4 The International Society on Thrombosis and Hemostasis (ISTH) developed a scoring system that makes use of laboratory tests, and a DIC score calculator. However, it is important to note, the presence of an underlying condition frequently associated with DIC is essential for the use of this diagnostic algorithm.4 Studies have reported the sensitivity of the DIC as 93%, and the specificity as 98%.4 As well, this scoring system has shown to be a strong predictor for mortality in sepsis. Similar scoring systems have been developed in other countries as well. Other conditions that may be ruled out by these tests include thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), immune thrombocytopenia (ITP), chronic liver disease, Heparin-induced thrombocytopenia (HIT), and Coumadin/Vitamin K deficiency.2

Correlation of Laboratory Tests

Thrombocytopenia is the most common laboratory finding in DIC, reported in 93% of cases.5 In many cases, the thrombocytopenia may not yet be severe; therefore, it is crucial to recognize a downward trend in platelet count despite a recent count in a normal range.5 Acute DIC typically presents with a rapid consumption of platelets and clotting factors, so that at the time of diagnosis, the platelet count may be less than 50 x 109/L, the PT and aPTT values are markedly prolonged.2 FDPs appear elevated, as they are released into circulation as the body attempts to break down clots. Fibrinogen levels may be decreased, as it is used up. Likewise, plasminogen and AT show a decrease.2 D-dimers appear increased as a result of fibrinolysis and excess plasmin.2 Red blood cells (RBCs) that are pushed through occluded vessels in the microvasculature become fragmented and will often appear on a peripheral blood smear as schistocytes.

Impact of Pathophysiology on Laboratory Results

DIC can present with a variable range of abnormal laboratory values. Additionally, the values may only represent a momentary glimpse into a rapidly changing systemic process and often requires frequent repeated testing.4 As well, patients with chronic DIC may present with only minimal abnormalities in laboratory tests. There is no single test that is a specific indicator for DIC. Rather, the use of combined tests such as platelet count, PT, aPTT, fibrinogen, and D-dimer, in conjunction with an underlying disorder, help guide management and treatment.

A diagnosis of DIC is reached only through a combination of the clinical impression in conjunction with laboratory abnormalities.5 The patient history plays a significant role in that DIC is always secondary to an underlying condition, therefore the clinical presentation of DIC will be the features of the primary disease.

Treatment

The treatment of DIC typically involves focusing on the underlying disorder, as manifestations of DIC will subside once the underlying condition is addressed. Management of DIC itself follows four basic features: monitoring vital signs, assessing and documenting the extent of thrombosis and hemorrhage, correcting hypovolemia, and administering basic hemostatic procedures when indicated.4 Therapeutic interventions are mostly supportive in nature, and may include transfusion of blood components including platelets and clotting factors. However, blood components should be reserved for those patients who have hemorrhage, require a surgical procedure, or pose a high risk for bleeding complications.2 The use of heparin is usually reserved for cases of chronic DIC, and should be provided to patients demonstrating widespread fibrin deposition without evidence of substantial hemorrhage.4 Generally, the use of antifibrinolytic drugs should be avoided, as they have been known to cause thrombotic complications, although these agents have proven useful in cases associated with AML-M3 and other types of cancer.4 In these cases, Heparin should always be administered with antifibrinolytic drugs so as to arrest any prothrombotic effects.4 In patients with trauma and massive blood loss, antifibrinolytics such as tranexamic acid has shown to be effective in reducing blood loss and improving survival.4 Only minimal cases have been reported to benefit from administration of activated PrC.

Patient Response to Treatment

Treatment decisions for this patient are based on clinical and laboratory evaluation of hemostasis. The patient is to avoid all invasive procedures such as IV-line placement and biopsies. Heparin, in this case, is contraindicated secondary to bleeding risk. The patient received FFP, platelets, and fibrinogen concentrate. The patient currently remains hemodynamically stable, however, if evidence of significant bleeding and hyperfibrinolysis remains, tranexamic acid may be administered in conjunction with heparin. Clinical and laboratory parameters will continue to be monitored every eight hours.  

Prognosis

The prognosis of patients with DIC depends on the severity of the coagulopathy, as well as the underlying condition that incited DIC. Generally, if the underlying condition is self-limiting or can be appropriately managed, DIC will disappear, and coagulation status will return to normal.4 Since DIC is a complex process, it is crucial to monitor the patient for clinical improvement or worsening, as well as to identify the early development of possible complications.

Treating the underlying AML-M3 is critical for the prognosis and management of this patient. The patient is encouraged to regularly consult with a hematologist for assistance with management of DIC, as chronic DIC may be treated on an outpatient basis after stabilization.4 Chronic DIC in patients with cancer may be managed with low molecular weight heparin or subcutaneous heparin. As well, other subspecialty consultations are necessary as indicated by the patient’s primary diagnosis.

Conclusion

DIC is not a specific illness, rather, it is a complication or an effect secondary to the progression of other underlying illnesses. It is associated with numerous clinical conditions, which involve excessive activation of the coagulation system.

Although interventions are supportive, they have been shown to be only partly effective. Since the clinical course of DIC ranges from asymptomatic to life-threatening, understanding its pathogenesis is vital for early recognition. Despite diagnostic criteria to aid physicians in the management of patients with DIC, newer laboratory markers and treatment modalities are warranted to improve patient outcome.

References

1 Levi M, Scully M. How I treat disseminated intravascular coagulation. Blood. 2018 Feb 22;131(8):845-54. doi: 10.1182/blood-2017-10-804096

2 Boral BM, Williams DJ, Boral LI. Disseminated intravascular coagulation. Am J of Clin Pathol. 2016 Dec;146(6):670-68. doi: 10.93/AJCP/AQW195

3 Medline Plus [Internet]. Bethesda (MD): National Library of Medicine (US); updated [date]. Disseminated intravascular coagulation; [updated 2022 Mar 21; reviewed 2019 Sep 24; cited 2022 Mar 30]; [about 2 p.] Available from: https://medlineplus.gov/ency/article/000573.htm

4 Levi MM, Schmaier AH. Medscape [Internet]. Emedicine; [2020 Dec 06; 2022 Apr 22]. Available from: https://emedicine.medscape.com/article/199627-overview

5 Thachill J. Disseminated intravascular coagulation: A practical approach. Anesthesiology. 2016 Jul;125(1): 230-6. doi: 10.1097/ALN.0000000000001123

6 Rodak BF, Fritsma MG, Fritsma GA. Normal hemostasis and coagulation. In: Hematology: Clinical principles and applications. St. Louis, MO: Elsevier Saunders; 2012. p. 626–44 

7 Hussain SA, Zafar A, Faisal H, Vasylyeva O, Imran F. Adenovirus-associated disseminated intravascular coagulation. Cureus. 2021 Mar 30;13(3)e14194. doi: 10.7759/cureus.14194 8 Onishi T, Ishihara T, Nogami K. Coagulation and fibrinolysis balance in disseminated intravascular coagulation. Pediatr Int. 2021 Nov;63(11):1311-18. doi: 10.1111/ped.14684

-Kaysi Bujniewicz, MLS(ASCP)CM graduated Magna Cum Laude from University of North Dakota School of Medicine with a Bachelor of Science in Medical Laboratory Science. She has worked in clinical laboratories for over eight years as a certified and licensed Medical Laboratory Technician and a Medical Laboratory Scientist. Although her true callings are in Immunohematology and Clinical Microbiology, she currently works as a Generalist.

Coagulation Case Study: 14 Year Old Female With a History of Bleeding Episodes

Case Study

A 14 year old female arrived at the emergency room with her mother and grandmother complaining of extremely heavy menstrual bleeding. Patient history reported by her mother included a history of “a bleeding problem” for which she had been treated a few times since age 4. Petechiae were noted on the girl’s abdomen, arms and thighs. There was no history of aspirin or other NSAID use. Blood work was ordered.

Patient results are shown in Table 1 below.

The mother called home to ask her husband for details and reported that her daughter had been diagnosed with Immune Thrombocytopenic Purpura (ITP) 10 years earlier but was not very clear on the treatments. She stated that other than frequent nose bleeds, some petechiae, and occasional bruising that the girl had seemed ok until she started menstruating. They had not seen the specialists in a number of years. Further questioning of the mother revealed that the parents had both immigrated from Iran with their families as infants. The patient was an only child. The grandmother reminisced about the village “in the old country” and mentioned that her daughter and son in law were related, the families being from the same village. When asked about any other family with bleeding disorders, the mother reported that neither she nor her husband had ever met any other relatives in Iran and were unaware of any bleeding tendencies in the family. The grandmother interjected that she did remember that several of her cousins and an uncle experienced frequent epistaxis.

The ER physician noted the normal PT/INR, APTT and slightly decreased platelet count but felt the extensive petechiae and hypermenorrhagia were out of proportion to these results. A manual differential was ordered. Differential results were within normal ranges, RBC morphology reported sight polychromasia and anisocytosis. Platelet estimate was slightly decreased with giant platelets noted. The physician suspected an inherited platelet disorder and the patient was referred to a hematologist for further workup.

Image 1. Giant platelets on peripheral blood smear.
Image 2. Giant platelets.

Discussion

I have written a few blogs about different thrombocytopenias. This case interested me because the patient was first diagnosed with ITP. ITP is an autoimmune bleeding disorder in which the immune system makes anti-platelet antibodies which bind to platelets and cause destruction. Even though the exact cause of ITP remains unknown, it is recognized that it can follow a viral infection or live vaccinations. In children this tends to be an acute disease which is self-limiting and self resolves in several weeks. However, in a small number of children, ITP may progress to a chronic ITP, as was thought to be the case in this patient.

A new hematologist saw the patient and reviewed the medical history. In this patient, the diagnosis of ITP had been followed for a short period of time in which the platelet count did not increase. She was treated with immunoglobulin. When her platelet count dropped below 30 x 103/μL, the patient was transfused several times. Early platelet transfusions increased her counts, but the patient became refractory and was then given HLA matched platelets, with some improvement. After a period of time, the patient did not return to the specialist and the parents described her condition as improved. However, as reported to the ER physician, she still experienced frequent epistaxis and other bleeding symptoms unrelated to accidental injury. The mild thrombocytopenia and giant platelets on the blood smear with normal PT and APTT in a patient with abnormal bruising or bleeding alerted the physician to the possibility of the diagnosis of Bernard Soulier Syndrome (BSS). The family history also suggested BSS.

The hematologist ordered further testing. Noted in the patients chart from 10 years ago was a prolonged bleeding time. This test was not repeated at this time because it has largely been replaced by platelet function analyzers (PFAs.) The PFA test analyzes platelet function by aspirating citrated blood through membranes to induce platelet adhesion and platelet plug formation. The test is first performed with a collogen and epinephrine membrane (Col/Epi). If the closure time is normal, platelet function can be considered normal. If the closure time with Col/Epi is increased, then the test is repeated with a collogen and ADP membrane (Col/ADP). A prolonged closure time with Col/Epi with normal Col/ADP closure time may indicate an aspirin induced platelet disorder, whereas an increased closure time with both membranes may indicate a platelet defect that is not aspirin related.3 The PFA closure times were increased in both the Epinephrine and ADP cartridges.

Platelet aggregation was normal with all agents except ristocetin. BSS can be differentiated from von Willebrand disease(vWD) by the addition of normal plasma to the ristocetin agglutination test. The addition of normal plasma adds vWF to the suspension, and in vWD the ristocetin agglutination is corrected. Agglutination with ristocetin requires vWF and GPIb/IX. Since GPIb/IX is absent or reduced in BSS, he ristocetin agglutination is not corrected in BSS, as seen in this patient.3 Flow cytometric analysis of platelet glycoproteins demonstrated reductions in CD42a (GpIX) and CD42b (Gp1bα).

Bernard Soulier syndrome (BSS), also known as Hemorrhagiparous thrombocytic dystrophy, was first described in 1948 as a bleeding disorder characterized by a prolonged bleeding time and giant platelets seen on a peripheral smear. It is an inherited platelet adhesion disorder caused by platelet glycoprotein (GP) deficiencies. The disorder is rare, affecting only about 1 in 1,000,000, though it is more common in families where parents are related. BSS is typically autosomal recessive, though a small number of cases have been found that are autosomal dominant. Most cases are diagnosed at a young age, with the autosomal dominant type often less severe and diagnosed later in life.1

Platelets are involved in primary hemostasis, the initial arrest of bleeding that occurs with vascular injury. As we know, platelets’ functions include adhesion and aggregation. Platelets first stick to the blood vessel wall (adhesion), followed by binding to each other (aggregation). In primary hemostasis, platelets first adhere to von Willebrand factor (vWF) which is bound to the subendothelial collogen fibers. This is followed by aggregation, a complex process that results in the formation of the platelet plug and the initial arrest of bleeding.. In BSS, platelet membrane GPs Ib, V and IX are missing, resulting from an inherited mutation in one of the genes that code for proteins in the complex. This affects the binding of the platelets to vWF, which subsequently interferes with primary hemostatic plug formation.4 If the platelets don’t adhere, aggregation is also affected.

Patient Results

In order to make a differential diagnosis of platelet function disorders, laboratory testing is necessary:

  • Tests of secondary hemostasis, PT and APTT, are normal in this patient so a disorder of primary hemostasis would be suspected.
  • In this patient, the platelet count was slightly decreased. In BSS, the platelet count is variable, from normal is moderately decreased, and can vary from time to time in the same patient.
  • Platelet adhesion tests (PFA) performed with both Col/Epi and Col/ADP were abnormal.
  • Light transmission aggregometry revealed platelet aggregation was normal with ADP, collogen and epinephrine. Aggregation with ristocetin was abnormal.
  • Giant platelets observed on peripheral smear
  • Flow cytometric analysis of platelet glycoproteins demonstrated reductions in CD42a (GpIX) and CD42b (Gp1bα).

Diagnosis: Bernard Soulier syndrome.

Conclusion

BSS is rare and is commonly mistaken for ITP. Reports have been published that analyze cases of BSS patients long treated as ITP. These misdiagnosed cases have been treated with immunoglobulins, steroids, IV anti-D, and other drugs used to treat refractory ITP. Splenectomies have even been reported in some cases. Platelet aggregation to ristocetin and flow cytometry have provided the correct diagnoses. Molecular studies can also be done to identify the abnormal genotype.2 Clues that can lead to a correct diagnosis are childhood ITP that does not spontaneously resolve and does not respond to treatments, other family members with bleeding problems or low platelet counts, platelet counts that are not low enough to explain bleeding or prolonged bleeding times, increased MPV and the presence of giant platelets on the peripheral smear.

This patient was diagnosed with ITP as a child, but treatments did not improve her platelets counts. She continued to have bleeding episodes which increased with the onset of menses. Her grandmother reports a history of bleeding tendencies in other family members. In addition, her parents are related. Her peripheral smears noted giant platelets. Laboratory tests confirmed a diagnosis of BSS.

Bernard Soulier syndrome (BSS) is a rare but important long-term bleeding disorder.

Patients do not require routine prophylactic treatment, so the management of BSS focuses on prophylactic treatment before certain procedures or after injuries. Patients should be advised not to take NSAIDS. The patient should be advised that treatment may be necessary prior to procedures or in response to common bleeding events such as bleeding gums, epistaxis, and menorrhagia. Antifibrinolytic therapy can be used in bleeding episodes. Platelet transfusions are considered for patients before surgery or if anti-fibrinolytics have failed. For severe cases, stem cell transplants have provided a cure. BSS may also be a candidate disorder for gene therapy in the future.1

References

  1. Grainger JD, Thachil J, Will AM. How we treat the platelet glycoprotein defects; Glanzmann thrombasthenia and Bernard Soulier syndrome in children and adults. Br J Haematol. 2018 Sep;182(5):621-632. doi: 10.1111/bjh.15409. Epub 2018 Aug 17. PMID: 30117143.
  2. Reisi N. Bernard-Soulier syndrome or idiopathic thrombocytopenic purpura: A case series. Caspian J Intern Med. 2020;11(1):105-109. doi:10.22088/cjim.11.1.105
  3. Perumal Thiagarajan, MD; Chief Editor: Srikanth Nagalla, MBBS, MS, FACP. https://www.medscape.com/answers/201722-90211/what-is-the-platelet-function-analyzer-100-pfa-100-and-how-is-it-used-in-the-workup-of-platelet-disorders
  4. Turgeon, Mary Louise. Clinical Hematology, Theory and Procedures. Fifth ed. 2012. Lippincott Williams and Wilkens. Baltimore.
Socha-small

-Becky Socha, MS, MLS(ASCP)CMBBCM 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 40 years and has taught as an adjunct faculty member at Merrimack College, UMass Lowell and Stevenson University for over 20 years.  She has worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. She currently works at Mercy Medical Center in Baltimore, Md. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

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.