Case Study Mini-Series; Diagnosis

The patient was diagnosed with subclinical DIC because complications from Acute Promyelocytic Leukemia (APL)

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The characteristic chromosomal translocation of Acute Promyelocytic Leukemia is the break and fusion of the PML gene located on chromosome 15 and the RARA gene located on chromosome 17. This results in a t(15;17) which is detectable in more than 90% of cases. The PML gene has a physiological role in apoptotic pathways and in genomic stability. The t(15;17) breakpoint in PML can occur in three different sites; bcr1 within intron 6, bcr2 within exon 6, and bcr3 within intron 3 . The RARA receptor is active in different variations within each tissue and is important for granulopoiesis. The PML-RARA fusion transcripts impair signaling which is mediated by RARA and interact with proteins that leads to the delocalization of normal PML from its nuclear structures known as NBs. It is in this way that the PML-RARA oncoprotein negatively acts on the normal physiology of the native PML protein. APL is a subtype of AML that has distinctive morphological, biological and clinical characteristics. It is classified as AML-M3 in the French-American-British (FAB) classification system. The cure rate for APL is ~80-90% for patients who survive induction therapy with ATRA. Before ATRA, the 10-day survival rate with treatment was 9.4%. A high blast count was significantly associated with hemorrhagic events and fatality within the first 10 days. A high blast count and thrombocytopenia was associated with death within 24 hours upon admission and treatment. APL predominantly affects a wide spectrum of individuals between the ages 20 and 59 with no gender discrimination. 10-15% of all AML diagnosed in adults is APL, although it can be seen in distinct populations in a higher percentage. 28.2% of all AML diagnosed in Brazil is APL, and 20% of all AML in Venezuela is APL.

APL presents as a bleeding diathesis and coagulopathy. The more common hypergranular variant of APL presents with leukopenia while the less common microgranular variant tends to be more aggressive and presents with leukocytosis. The malignant promyelocytes have specific properties that interact with the host cells. Maligant APL cells express tumor associated procoagulants; Tissue factor (TF) and cancer procoagulant (CP). Tissue factor is an activator of coagulation and the relative expression is elevated significantly in patients with APL.

APL is characterized as a hyperfibrinolysis state. Fibrinolysis is normally activated by thrombin as the fibrin clot develops and coagulation comes to an end. Malignant promyelocytes highly express annexin-II. Annexin-II is a protein receptor that has a strong affinity to plasminogen and tissue-type plasminogen activator (tPA) which results in strong yield of plasmin which initiates fibrinolysis. Annexin-II is highly expressed in the cerebral microvascular endothelial cells explaining the high prevalence of intracerebral hemorrhage in patients with APL. Cytokine release of IL-1B and TNFa by malignant promyelocytes upregulate apoptosis and upregulate the expression of tissue factor on endothelial cells. It is also common for the cytokines to cause loss of the anti-coagulant cofactor thrombomodulin. These various factors lead to APL-associated coagulopathy commonly seen.

Patients with APL present low fibrinogen levels, low platelet count, and an elevated PT-INR, aPTT, and D-dimer. In DIC secondary to APL, fibrinogen survival is markedly decreased due to rapid consumption and the liver can’t produce the product fast enough. Sometimes more specialized tests are needed to diagnose the coagulopathy in APL. Levels of thrombin-antithrombin complex (TAT), prothrombin fragment 1 and 2, and fibrinopeptide A are all increased and all indicate coagulation activation. Decreased levels of plasminogen, and a-2-antiplasmin further support the hyperfibrinolysis state. Sometimes it is helpful to further evaluate the coagulation process and its components. Protein C and antithrombin III are synthesized in the liver and are relatively normal in APL associated coagulopathy unless the maligancy is accompanied by hepatic dysfunction.

The next installment of the mini-series will focus on the key points of what lead to the diagnosis, what I look for as a medical laboratory professional in aiding the doctor in the diagnosis, and how to treat appropriately.

 

-Caleb

 

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37-year-old South American Male Case Study Mini-Series

The purpose of this mini-series is to get in the mind of a treating physician when a patient such as this presents to the clinic or the ED in this case. The first part of this series is the introduction of the case with case history and initial lab testing. Please don’t hesitate to leave comments on what you think the diagnosis is and what other confirmatory tests need to be done if any as well as what treatment should consist of. This is mean’t to stimulate a discussion and there are no wrong answers. I am in no way a physician or at that level or have that education. I am a student with a passion for molecular diagnostics and creating these cases is a good way for me to practice real life scenarios through careful and diligent research as well as help others who think the same way. This case is no way real and all lab values are made up to the best of my knowledge. If anything is incorrect please do not hesitate to email me or  leave a comment.

CASE:

A 37-year-old South American male presented to his annual physical with his primary care physician with general fatigue, decreased appetite and weight loss over the past three weeks. The patient mentioned to his physician that he has had multiple nosebleeds throughout the last few weeks, an occurrence of multiple a week. The patients past medical history is unremarkable. No family history of bleeding tendencies. He is not taking any prescription medication and denies use of recreational drugs and only social use of alcohol. His physician ordered a CBC and a prothrombin time/activated partial thromboplastin time (PT/aPTT). Results are in table 1.

Two days later the patient presented to the emergency room with fever and heavy fatigue, he explained to the attending that it has been hard to do anything the last few days, and has been bed-ridden. Physical exam revealed bilateral bruising on the upper arms and forearms with purpura and petechiae. The attending physician ordered a full coagulation panel, platelet function tests (Ristocetin cofactor assay), bleeding time test for vWD, and full CBC with peripheral blood smear analysis. Results are summarized in table 2.

Later that evening the patient developed a high fever, and back/flank pain and was moved to the ICU. Blood cultures, CRP and a procalcitonin was ordered, results are in table 3.

Positive cultures for Staphylococcus aureus were found after 48-96 hours and the patient was started on a course of vancomycin and monitored closely.

Patient results indicated he was pancytopenic with a hemoglobin of 9.7 g/dL (Ref. 13.5-18.0 g/dL) and RBC count of 3.7×10^3/uL (Ref. 4.20-6.00×10^6 uL) with severe thrombocytopenia at 37×10^3/uL (Ref. 150-450×10^3/uL).

Initial coagulation results revealed significantly elevated PT and aPTT. The bleeding time test along with the results from the RCO indicate platelet dysfunction or acquired inhibition of platelets by accelerated destruction. Platelet aggregation studies were normal. RCO studies indicate factor VIII inhibition or consumption.

The peripheral blood smear confirmed leukopenia and thrombocytopenia and revealed abnormal promyelocytes with abundant azurophilic granulation and multiple auer rods in bundles. RBC morphology showed schistocytes and fragmented cells.

The attending followed up by ordering a complete fibrinogen, D-dimer and a plasminogen panel. Results are in table 4.

The significantly elevated D-dimer, elevation in t-PA and u-PA in combination with the significant decrease in fibrinogen, and plasminogen levels indicates primary hyperfibrinolysis.

The attending sent a blood sample to the Blood Bank laboratory and asked for units of packed red cells, platelets, and fresh frozen plasma (FFP) to be transfused. With the additional blood components, the patient was able to regain control over the thrombocytopenia, hemoglobin, fibrinogen and coagulation factor levels.

A bone marrow aspirate was ordered including cytology, cytochemistry, immunophenotyping, FISH (Fluorescence in situ hybridization), cytogenetics (chromosomal analysis and FISH) and RT-PCR for PML/RARA quantification of transcripts. The attending started the patient on all-trans retinoic acid (ATRA) as induction therapy.

FISH revealed the PML-RARA fusion gene present which was later quantified and confirmed by RT-PCR. PCR sequencing revealed a bcr-3 PML-breakpoint. Chromosomal analysis of the bone marrow identified a t(15;17) classic translocation. Cytochemistry revealed intensely positive reacting cells to myeloperoxidase and Sudan black B. Immunophenotyping results are in table 5.

Table 1:

RBC: 4.10×10^6/uL             4.20-6.00×10^6/uL          

HGB: 12.9 g/dL                    13.5-18.0 g/dL        

HCT: 38.7%                          40-54%

MCV: 88 fL                            80-100 fL

MCH: 33.2 pg                        26-34 pg

MCHC: 32.3 g/dL                  32-36 g/dL

RDW: 13.5%                         11.5-14.5%

RETIC: 0.8%                          0.5-2.5%

NRBC: 0/100 WBC               0

WBC: 6.3×10^3/uL              3.6-10.6×10^3/uL

NEUT: 3.6×10^3/uL             1.7-7.5×10^3/uL

LYMPH: 1.9×10^3/uL          1.0-3.2×10^3/uL

MONO: 0.7×10^3/uL           0.1-1.3×10^3/uL

EO: 0.1×10^3/uL                  0.0-0.3×10^3/uL

BASO: 0                                 0.0-0.2×10^3/uL

PLT: 111×10^3/uL              150-450×10^3/uL

MPV: 7.3 fL                           7.0-12.0 fL

 

PT: 21 seconds                    11-14 seconds

aPTT: 37 seconds               25-35 seconds

Table 2:

RBC: 3.7×10^3/uL              4.20-6.00×10^3/uL

HGB: 9.7 g/dL                     13.5-18.0 g/dL                    

HCT: 28.9%                          40-54%

MCV: 71 fL                            80-100 fL

MCH: 31.8 pg                       26-34 pg

MCHC: 33.1 g/dL                 32-36 g/dL

RDW: 15.1%                        11.5-14.5%

RETIC: 2.3%                         0.5-2.5%

NRBC: 0/100 WBC               0

WBC: 2.5×10^3/uL             3.6-10.6×10^3/uL

NEUT: 1.3×10^3/uL           1.7-7.5×10^3/uL    

LYMPH: 0.7×10^3/uL         1.0-3.2×10^3/uL

MONO: 0.3×10^3/uL          0.1-1.3×10^3/uL

EO: 0.1×10^3/uL                 0.0-0.2×10^3/uL

BASO: 0.1×10^3/uL            0.0-0.3×10^3/uL

PLT: 37×10^3/uL               150-450×10^3/uL

MPV: 19.3 fL                       7.0-12.0 fL

 

Myeloblasts: 7%                  0%

Promyelocytes: 54%         0%

Myelocytes: 3%                    0%

Metamyelocytes: 5%           0%

Bands: 0%                             0%

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PT: 33 seconds                   11-14 seconds

aPTT: 63 seconds              25-35 seconds

BT: 13 minutes                   1-9 minutes

RCO: 30%                              50-150%

Platelet aggregation studies: Normal

Table 3:

Blood Cultures: POS Staph aureus          NEG

Procalcitonin: 0.25 ng/mL                        <0.15 ng/mL

CRP: 23 mg/L                                                0-10 mg/L

Table 4:

Fibrinogen: 67 mg/dL

Plasminogen: Reduced

a2-Antiplasmin: Reduced

t-PA: Elevated

u-PA: Elevated

D-Dimer: >19,000 ng/mL    

Table 5:

CD2                NEG

CD4                NEG

CD13              POS

CD14              NEG

CD16              NEG

CD19              NEG

CD33              POS

CD34              NEG

CD45              POS

CD56              NEG

CD64              POS

CD117            POS

HLA-DR         NEG

 

-Caleb