December 2025: The Final Chapter
by Donna Castellone • December 15, 2025
I was asked by ANIARA DIAGNOSTICA to support their commitment to continuing education in the field of coagulation almost 20 years ago. They asked me to provide readers with up-to-date information on current issues in coagulation. Hence this blog was born! I wanted to be able to provide a platform that gave readers a chance to be informed about available seminars that were taking place, which led to What's Happening in Coagulation. Next, came What's New in Coagulation which was based on what was new in coagulation as well as findings from clinical trials. I eventually added the Journal Club which were abstracts of new and relevant articles in the field of laboratory medicine. Finally, to round it out, came Coagulation Corner where each month I chose a topic from a relevant discovery, or summary of a conference attended, or even just the application of coagulation in our everyday life. I hope that I have enriched your knowledge, stimulated thoughts and helped solve coagulation problems. I hope that it has served your coagulation laboratory and knowledge well.
So it is with very mixed emotions that I am going to step away from this endeavor and concentrate on travel, painting, reading and my family. My favorite post each year was my holiday post which has included in the past "Twas the Night before Christmas in the Coagulation Laboratory", Elf on the Shelf "The Case of the Elevated D-dimer in Santa" and, of course, "Christmas Disease in the Coagulation Laboratory". My one final post will be:
A COAGULATION CORNER: PAST, PRESENT AND FUTURE
Dr. Scrooge is at it again- no matter the time of day, the day of the week, or holiday. Not a request but an order for a full coagulation workup on his patient. The doctor calls the Special Coagulation laboratory to initiate the request: PT/INR, aPTT, Platelet Count, Fibrinogen, D-dimer, Protein C, Protein S, Fibrinogen, Factors VIII and IX assays. Lupus workup, AT, and how about a HIT assay. Results are expected as soon as possible. Oh, and please add on a bleeding time.
Coagulation laboratory PAST: 40 years ago
A special coagulation technologist is called to the floor and test requests are on a paper requisition. The technologist prepares the tubes: 2 tubes for PT/aPTT and Fibrinogen, 2 tubes for Protein C and S, 2 tubes for FVIII and IX, 2 tubes for a Lupus workup, a purple top tube for the platelet count, and a red top tube for the HIT assay. Well, this is some work up- and what is a D-dimer? We could manage an FDP, but it needs to be drawn in that special tube. It has a yellow label and trypsin inhibitor and Bothrops atrox venom. Get out the slide with the wells and check for agglutination.
Platelet count will be performed manually on a hemocytometer chamber in duplicate after the RBC are lysed with a dilutent. Count the platelets in the grid.
Okay, so anticoagulation is either warfarin or unfractionated heparin.
For the bleeding time, the patent is scheduled and a tech from the special coagulation laboratory is sent to perform the test. A blood pressure cuff is placed on the arm and a cuff pressure of 40 mm Hg. The bleeding time lancet is placed on the forearm and a standardized cut is made. A stopwatch is started and every 30 seconds the drop is “wicked” with filter paper. The time is recorded as to when the cut stops bleeding. This is a test to analyze platelet function. It takes about 1 hour for the tech to leave the lab and perform the test. If they can find the patient, and they are available!
The PT (there is no INR) as well as the aPTT will be run in duplicate on a semi-automated analyzer. The aPTT is abnormal, so it then will be repeated by the tilt tube method to visualize the clot. PT reagent is rabbit brain thromboplastin. APTT has an activator, phospholipid, and added calcium. Results are ready in 3 hours.
Fibrinogen is performed on a fibrometer and calculated off a standard curve. The controls do not come out, so everything needs to be repeated.
Protein C and Protein S antigen will be performed by electrophoresis and take about 3 days between preparing the gel, running the gel, drying and staining. Hopefully the standard curve and controls come in so the patient results can be read off the graph that will have to be constructed. AT will be performed via radial immuno diffusion which can be read with a loop after 72 hours.
HIT will be sent to a special center that performs Serotonin release assay. Should be back in a week or two.
Lupus testing: A mixing study is done, both the immediate and incubated mix. The dilute russell viper venom test is done on the fibrometer in duplicate.
Factor VIII and Factor IX:
These will take all day!
The deficient plasma for FVIII and FIX is finally able to be purchased via a company and doesn’t have to be made. You would previously need two reagents, aged serum and BaSO4 or Al(OH)3 absorbed plasma. To prepare aged serum, collect blood, allow to clot, separate the serum and incubate at 37° C for 24 h to destroy factors V and VIII. Aliquot and freeze. Aged serum provides factors VII, IX, X, XI and XII. If prepared correctly, aged serum will generate no clot in either the prothrombin time (PT) or partial thromboplastin time (PTT) assay. This would be used to test for Factor VIII deficiencies.
For absorbed plasma, collect fresh anticoagulated blood, separate, and add Amphogel (Al(OH)3) or dry BaSO4 to the plasma for a few hours to absorb the vitamin K-dependent factors. Mix occasionally, centrifuge, aliquot, and freeze. Absorbed plasma provides fibrinogen and factors V, VIII, IX and XII.
First a standard curve will have to be made for each factor. Manual dilutions, 7 of them (made independently- not serially) will have to be run in duplicate, then averaged (as long as they are within acceptable limits), then the seconds of each dilution will be graphed against the value of the dilution of the standard. A low and normal control will be run in duplicate and if that works the patient plasma will then have 5 dilutions made independently and then run in duplicate. The seconds will be read off the standard curve and multiplied by the appropriate dilution. Factors are finally done- took a full shift.
Now Dr. Scrooge has pieces of a profile, after a full week still waiting for some results. The patient has already been discharged and he doesn’t really remember the case anyway.
Coagulation Laboratory; Present Day:
Dr. Scrooge enters a Screening panel with platelet count, thrombophilia work up, including FVIII and FIX genetic testing FVL, MTHFR and Prothrombin Mutation. HIT testing. Requests a PFA to evaluate primary hemostasis. Sends down 4 TUBES. EPIC shows the patient not on any anticoagulant at time of draw. Testing is based on the laboratory coagulation cascade, however the cell-based model of coagulation has been accepted to capture the accurate in vivo interplay of plasmatic coagulation factors as well as platelets and tissue factor (TF)-bearing cells.
PT/INR, aPTT, D-dimer, fibrinogen and FVIII and FIX are completed within 2 hours in the routine laboratory on 1 tube. Total laboratory automation with interfacing of hemostasis analyzers onto automated laboratory delivery lines perform the testing.
Recombinant human TF-based allowing further standardization of PT testing having the recommendation for an ISI close to 1.0. This is used in the calculation of the INR which standardizes the PT result regardless of the reagent instrument combination. Platelet count fails delta check, has dropped by 60% from yesterday, needs to be confirmed via slide, but floor has preliminary result.
The PFA is performed. The PFA-100 is a system for analyzing platelet function in which citrated whole blood is aspirated at high shear rates through disposable cartridges containing an aperture within a membrane coated with either collagen and epinephrine (CEPI) or collagen and ADP (CADP). These agonists induce platelet adhesion, activation and aggregation leading to rapid occlusion of the aperture and cessation of blood flow termed the closure time (CT). The PFA-100 has a high negative predictive value i.e., if the PFA-100 gives a normal result then with some exceptions platelet functional defects can be ruled out. The result is normal and completed within 30 minutes.
The thrombophilia workup PC, PS, AT, functional and antigen and Lupus testing (mixing study, DRVV and Hexagonal phase, are being run simultaneously in the Special Coagulation Laboratory, as well as the HIT assay performed by ELISA testing.
The molecular laboratory will perform genetic testing tomorrow. All results completed in two days. Dr. Scrooge almost is happy.
Coagulation of the Future:
Well, here I am stumped! I have seen so many changes in coagulation throughout the years. With the advances in molecular testing as well as the development of new anticoagulants and replacement therapy, testing is evolving to keep up with all the advances. It is constantly a moving target. Gene therapy has made incredible breakthroughs in hemophilia A and B. Not anything I thought I would see in my lifetime. Do I think that testing will be performed on a chip with a drop of blood? I have learned to never say never, but I hope if it does come to that, it is based on science and not some person who doesn't understand the first thing about laboratory testing. What about artificial intelligence? Will we just plug in symptoms and the required testing be performed? Will we lose our gut feeling when something doesn't sit quite right? I hope not. AI is here to stay, but I also believe that we will need that investigation and intuition.
Conclusion:
So here is my Coagulation laboratory Past, Present and Future. I have encountered one or two Dr. Scrooges throughout my career, but by far I have encountered many more dedicated, smart and caring physicians. I have worked in several coagulation laboratories and learned from the best. I enjoyed writing this blog and hope I have provided you with good information through the years. So to all my Clotter friends and Aniara, I thank you from the bottom of my heart and I hope that I have answered some of your coagulation questions, enhanced your knowledge and challenged you to ask difficult questions and always think of your patients! It has been an honor and a privilege!
Thank you so much,
Donna