Education Case: Hereditary Thrombophilia With Double Heterozygous Factor V Leiden and Factor II c.*97G>A Mutations

Abstract

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.¹.

Keywords: Factor V Leiden; coagulation cascade; coagulation disorders; hematopathology; hemostasis; inherited thrombophilia; organ system pathology; pathology competencies.

Figure 1.

The 2 arms of the classic coagulation cascade, the tissue factor pathway (also known as the extrinsic pathway) and the contact activation pathway (also known as the intrinsic pathway), each consisting of different factors that both activate the common pathway (factor X, thrombin, and fibrin), leading to cross-linking of fibrin and formation of the hemostatic plug. It is worth mentioning that this classic cascade represents mostly in vitro events that is important for laboratory testing (PT, aPTT, and TT) and does not entirely represent the in vivo (physiological) coagulation cascade.

Figure 2.

Each assay includes a size ladder (column 1) and 5 other columns (columns 2-6) in which different samples are added to reaction tubes. The size ladder represents the size of the migrating DNA fragments. Each one of the columns has 2 subcolumns in which the reactions occur: the wild type (wt) and the mutant types. In each column, the reagents are reacted with the 3 added primers generating different bands (B1, B2, and B3). The first band (B1) at 250 bp represents an amplification control primer. The second band (B2) at 340 bp represents the factor II mutation-specific DNA primer. The third band (B3) at 432 bp represents the factor V Leiden-specific DNA primer. The presence of migrating DNA fragments confirms the presence of the DNA sequence of interest. In column 2, a normal control (sample from healthy patient with no FVL or factor II mutations) is added to the reaction tube. Note the presence of 2 bands (B2 and B3) in the wt tube and the absence of those bands in the mutant reaction tube. The absence of both the factor V–specific (432 bp, B3) and the factor II–specific (340 bp, B2) bands in the wt reaction tube A, or the absence of the amplification control (250 bp, B1) in the mutant reaction, suggests either lack of template or polymerase chain reaction (PCR) inhibition and invalidates the run. In tubes 2 to 4, different controls are added as follows: normal control (column 2), heterozygous positive control samples for FVL (column 3), and heterozygous positive factor II (column 4). Column 5 include the test reagents with no added DNA sample, therefore, no bands are seen as there is no PCR reaction. Column 6 is the actual patient sample; note the presence of all 3 band (B1, B2, and B3) in the mutant reaction tube and absence of those bands in the wt tube.