Platelet Information and Courses from MediaLab, Inc.

A 63 year old man was seen in the emergency room with the complaints of sudden onset of fever, chills, and abdominal pain, accompanied by mild diarrhea. The blood pressure was 140/84, the pulse rate 82/minute, and the body temperature 39.8C. A blood sample was drawn for a complete blood count, and a blood culture.A second blood culture was drawn from the opposite arm, with 10 ml of blood being placed into each an aerobic and an anaerobic bottle, following customary practice.The complete blood count revealed a hemoglobin of 15.8 mg/dl, a hematocrit of 45%, and a white blood count of 4.2/L. The neutrophils were 39%, lymphocytes 45%, monocytes 10%, eosinophils 4% and basophils 2%. The platelet count was 255/L. The patient was admitted to the hospital for further work-up and empiric antibiotic therapy.Within 24 hours after admission, the body temperature had decreased to 38.2C, although the mild diarrhea persisted.A stool toxin test for Clostridium difficile was negative and neither enteric pathogens nor Campylobacter species were recovered in stool culture after 24 hours incubation. Fecal neutrophils were not seen on direct examination. The anaerobic blood culture became positive 36 hours after inoculation.

Vessel size as related to time required for clotting to occur, amount of products used (platelets and clotting factors), and size of the corresponding bleed.

Our blood circulates freely through undamaged, intact vessels. The design of the vasculature, or blood vessels, is such that the walls of the vessels are chemically inert to both coagulation factors and platelets under normal conditions. Damage to a vessel breaks that inert epithelial lining, exposing the subendothelium and collagen, and releasing chemical signals that trigger subsequent hemostatic mechanisms.

As discussed earlier, a break in the vessel endothelium leads to exposure of collagen and the vessel's subendothelial surface. Ruptured endothelial cells leak ADP and Serotonin, which are the chemical triggers that induce platelet adhesion, the next step in the sequence of hemostatic events. Circulating platelets are drawn to the area by those liberated chemical signals, and begin to physically attach themselves to the rough, damaged surfaces of the breach. As platelets continue to arrive and bind to the exposed collagen and basement membrane, a rudimentary barrier begins to form, as the platelets themselves serve to fill in the breached vessel wall. Platelets possess an inherent “sticky” property which enables them to adhere to one another, and not just to the damaged vessel endothelium. The process by which platelets bind to one another is referred to as platelet aggregation, and is vital because it allows for a platelet plug to be formed. The platelet plug is the structure responsible for plugging the hole in the vessel wall.

Platelets play a significant role in primary hemostasis, as they are the “bricks”, or building blocks of the developing platelet plug, the forerunner to the end stage fibrin clot. Platelets have inherent adhesive properties which are essential for adherence to the site of vascular damage, and for binding to one another in aggregation activities. Platelets must be present in sufficient number, and be functionally active for optimal clotting to occur. Platelet functionality tends to be more crucial than the number of platelets available, however, as patients with lower platelet counts can still clot relatively effectively as compared to those patients with intrinsic platelet defects.

Kinetic Processes Specific to Platelets. Adhesion – When platelets adhere to exposed collagen, they take on a characteristic “spiny” shape. Their inherent stickiness, and the aforementioned spiny shape serve to compliment each other during this process. Von Willebrands Factor (vWF) is absorbed by surface receptors on both the platelet and exposed subendothelial tissue, thereby linking the platelets to the tissue. Release – This process occurs prior to aggregation. Platelets dump the contents of their granules (ADP, Serotonin, & Calcium), which aids the upcoming aggregation process by acting as a chemical signal. Aggregation – Platelets physically bind to each other, not just to the exposed subendothelial walls and collagen of the breached vessel. Platelet aggregation requires sufficient chemical signal stimulation. Stabilization (technically part of secondary hemostasis as fibrin is a product of secondary hemostasis)– This process strengthens the platelet plug with the addition of interwoven fibrin strands, ultimately producing a fibrin clot. The durable fibrin clot is the ultimate goal of hemostatic processes.

In summation, we have covered the following sequence of events which comprise primary hemostasis. The process begins with damage to a vessel wall, as blood flows outside the vasculature. The body responds with vasoconstriction, decreasing blood flow to the affected area. Platelets begin sticking to the damaged vessel walls. As the platelets stick, they release chemicals which signal other platelets to respond. As other platelets arrive, they begin sticking to one another, clumping together, forming a plug to fill in the breach. This plug, while strong, is a temporary fix, and must be reinforced with fibrin strands to effectively fill the breach during the vessel repair process. Construction of the fibrin strands occurs during secondary hemostasis, our next topic to be covered.

Activated factor X (Xa), in the presence of factor V (necessary cofactor), ionized calcium, and platelet factor act to convert prothrombin (precursor) to its active form, thrombin, by cleaving the prothrombin molecule.

A platelet function assay (PFA) is a screening test for the evaluation of platelets/primary hemostasis. Common clinical applications include the following: Preoperative evaluation of platelet function Determining the presence of drug-induced platelet dysfunction Determining platelet functionality in high-risk pregnancy Evaluation of patients with suspected inherited or acquired platelet disorders such as von Willebrand disease Evaluation of a bleeding patientA PFA instrument is able to differentiate between drug-induced platelet defects and other platelet defects. PFA tests are superior to the bleeding time test. The bleeding time is often not reproducible and, in spite of attempts at standardization, remains prone to variations in test results between persons performing the test. It is also relatively insensitive to platelet function. The bleeding time cannot be used to identify patients who may have recently ingested aspirin or non-steroidal anti-inflammatory drugs or patients who may have a platelet defect attributable to these drugs. The bleeding time is used to assess platelet function, but may be affected by platelet quantity. NOTE: Aspirin, and some other drugs, may falsely prolong bleeding times. Patients must be asked about aspirin use, and be aspirin free for 7-10 days prior to testing, for valid results.

Von Willebrands Disease is a platelet disorder. This disorder is characterized by a functional defect in Von Willebrands factor (vWF) itself. This disease often clinically manifests with a concurrent deficiency of factor VIII, but will present with a normal platelet count. As far as genetics and inheritance, both men and women are affected equally. Von Willebrands factor is essential for platelet binding, therefore, a defect in vWF causes impaired platelet adhesion and aggregation. The treatment of Von Willebrands Disease involves the administration cryoprecipitate, as it is rich in vWF.

Bernard-Soulier Syndrome is a genetic platelet disorder characterized by abnormal platelet function tests, unusually large platelets, and a moderate decrease in platelet count. Clinically, patients present with mucotaneous bleeding of varying severity, as well as having gingival bleeds, epistaxis, purpura, and gastrointestinal hemorrhaging. Treatment can range from the administration of iron supplements up to red cell replacement therapy if the episodic bleeding is severe enough to warrant it.

Before learning to examine bone marrow microscopically, it is important to understand the basic structure and function of the bone marrow. The bone marrow is one of the largest organs in the body. The normal adult marrow on a daily basis produces approximately 2.5 billion red cells, 2.5 billion platelets and 1.5 billion granulocytes per kilogram of body weight. The main function of this organ is the formation and development of blood cells. Hematopoiesis begins in the yolk sac in the first weeks of embryonic life; stem cells from the yolk sac travel first to the liver and then to the spleen. These organs are the only blood forming sites during the first three months of fetal life. At the beginning of the fourth month the bone marrow begins its life-long function of cell production.

This field under high magnification shows an increased number of megakaryocytes (megakaryocytic hyperplasia). This patient had thrombotic thrombocytopenic purpura. He was therefore consuming increased numbers of platelets, and his bone marrow was responding by increasing the number of megakaryocytes, which of course break up into platelets.

Another example of a meta-megakaryocyte showing many of the platelets breaking away from the nucleus. Some are shown by the arrow.

It is also important to examine the morphology of platelets. One megakaryocyte shows a single nucleus surrounded by cytoplasm which will eventually break off to form platelets. The other one at the arrow shows a lobated nucleus which has divided several times; the large amount of cytoplasm surrounding this nucleus means that this cell will be able to produce more platelets. In general, as the megakaryocyte gets older, it forms more nuclear lobes, more cytoplasm and therefore is able to produce more platelets.

It is also important to note that in addition to red cells, ABO antigenic determinants (epitopes) are found in many tissues, body fluids, and other cells including endothelial cells and platelets. Because ABO antigens are so widely expressed, ABO antigens are also a major consideration in solid organ and bone marrow transplants.

Occasionally they occur in clumps, particularly if the film was made from capillary blood.

Platelets stain light blue to purple in color, and are very granular.

Platelets are derived from the cytoplasm of megakaryocytes, giant cells in the bone marrow. At any given time, two thirds of the total platelets are in the circulation and one third are present in the spleen. In persons with enlarged spleens 80-90% of the platelets are in the spleen resulting in a decreased concentration of circulating platelets. In individuals who have had a splenectomy all of the platelets will be in the circulating blood. The life span of the platelet is 8-10 days.

Their shape varies greatly, but they are usually round, oval or rod-shaped.

Platelets function both mechanically and biochemically in the process of hemostasis. When injury to a blood vessel occurs, platelets aggregate forming a plug which helps to stop the flow of blood. They release certain substances, among them serotonin and Platelet Factor 3. Serotonin causes the blood vessels in the area to constrict, thereby further stopping the flow of blood. Platelet Factor 3 catalyzes the coagulation reaction whereby a fibrin clot is formed, completing the seal. Platelets also maintain the integrity (leak-free) state of blood vessels.

The third group of formed elements in normal peripheral blood is made up of thrombocytes (platelets). Although platelets don't look very impressive, their role in the process of hemostasis is critical. Platelets are the small granular bodies shown with the arrows in this Wright stained smear.

N:C Ratio - Nuclear: cytoplasmic Ratio - The ratio of nuclear volume to cytoplasmic volume within any one cell.Neoplasm - Any new and abnormal growth, such as a tumor.Neutrophilic Granules - Specific granules present in the cytoplasm of neutrophils. These granules resemble pencil stippling and stain a lilac color due to their affinity for both basic and acid dyes.Phagocyte - Any cell that ingests microorganisms or other cells and foreign particles.Phagocytosis - The ingestion and destruction of microorganisms or other foreign particles.Plasma - The fluid portion of blood in which the various blood cells are suspended.PF3 (platelet Factor 3) - A lipoprotein component of the platelet membrane; functions as a surface catalyst during blood coagulation.Pseudopod - A temporary protrusion of the cytoplasm of a cell.Refractile - Capable of refracting or changing the direction of light.Senescence - The process or condition of growing old.Serotonin - A constituent of blood platelets and other cells and organs; induces constriction of the blood vessels.Specific Granules - Granules found in cells of the more mature stages of the granulocytic series. They have distinct staining reactions which differ with each type of granulocyte.T-cell - Thymus derived lymphocyte which mediates cellular immunity.Thrombocyte (Platelet) - A circular or oval disk found in the blood; concerned with hemostasis.Thymus - A ductless gland-like body situated in the anterior mediastinal cavity; reaches its maximum development during the early years of childhood.Vacuole - Any small space or cavity formed in the cytotoplasm of a cell.

When drawing blood into evacuated tubes, it is best to draw the tiger top and red top tubes first, so that tissue fluids and fragments that cause blood to clot are retained in a tube which will clot anyway. Such tissue fluids and fragments are most likely to be present in the first tube to be drawn. If tissue fluids and fragments are present in a light blue top tube used for coagulation tests, they will interfere with the test results. However, recent studies have shown that accurate coagulation results may be obtained from the first tube drawn. It is nevertheless recommended that tiger and red top tubes be drawn first when using the evacuated tube system.If blood had been drawn first into a syringe, the order in which the blood is expelled from the syringe into the tubes would be different. Since no anticoagulant is present in the syringe the blood must first be expelled into the light blue top tube, then the lavender top tube, and finally the tiger top tube. This is so that the blood is promptly mixed with the anticoagulants present within the light blue and lavender top tubes before clotting begins. Clots in a lavender top tube are likely to cause a falsely low platelet count.Relevant topics:Lavender top tubes, Light blue top tubes

Bleeding time is a test designed to measure mainly platelet function.It is performed by making a small incision in the forearm, and measuring the time it takes for the incision stops bleeding.

When a blood sample is left standing without anticoagulant, it forms a coagulum or blood clot. The clot contains coagulation proteins, platelets, and entrapped red and white blood cells.

Circulating whole blood is a mixture of: Plasma (which contains fluid, proteins, and lipids), and Formed elements, consisting of red cells, white cells, and platelets.

Formed elements are the cells suspended in the blood. They include: Red blood cellsWhite blood cells Platelets

The peripheral smear illustrated in the photograph was obtained from a patient with a recent renal transplant. The patient developed a rash, accompanied by nausea and diarrhea. Graft vs. host disease was clinically suspected. The peripheral smear findings are consistent with that diagnosis. The presence of spherocytes suggests a hemolytic process which is supported by the presence of nucleated RBCs. A few scattered schistocytes and the decrease of platelets suggests DIC. The presence of target cells presents the possibility of associated liver disease. Additional tests, particularly coagulation studies, should confirm the diagnosis of microangiopathic hemolytic anemia.

Keratocytes are cells which have been damaged due to contact with fibrin strands. Since the cut is incomplete, however, no hemoglobin has been lost from the cell. Keratocytes have one or two projections which may vary in length from a short point to a longer string. These projections are formed when red cells come in contact with fibrin strands during circulation. Two keratocytes can be seen in this field. The one in the center to the left of the platelet has a lightly-stained projection. The keratocyte in the lower part of the screen is more pronounced. A knizocyte can be seen in the upper left portion of the field.

Another example of a May-Hegglin-type body. This smear was from a case of pseudo May-Hegglin caused by drugs. Bizarre appearing platelets can also be seen in cases of pseudo May-Hegglin.

May-Hegglin anomaly is an autosomal dominant condition characterized by the presence of pale blue inclusions in neutrophils, giant platelets, and sometimes thrombocytopenia. Inclusions can also occur in eosinophils, basophils, and monocytes. May-Hegglin inclusions resemble Dohle bodies but are larger and more prominent. Acquired forms of this anomaly may also occur as a result of the use of cytotoxic drugs. May-Hegglin bodies seen under these circumstances are considered pathological.

The findings in the photograph from a peripheral blood smear would elicit a report comment of "increased platelets" of some high magnitude, such as "marked" or "4+". Estimates of platelet counts from review of a peripheral blood should be made on each smear examined. This provides a simple estimate of "high" or "low" or corroborates the value generated from an electronic cell counter. A formula for estimating platelet counts must be established in each laboratory. Following is a guideline: 5/oil power field (OPF) = 100,000/cumm; each platelet thereafter = 10,000/cumm. Thus, if an average of 10 platelets/OPF are observed, the estimated platelet count is 150,000.cumm. Such a counting scheme for platelets when clustered as in the photograph is probably not needed, as there are more than 100 platelets in the field. This translates into a platelet count of 1 million/cumm or more. This peripheral smear observation, however, would serve to corroborate an electronic platelet count of 1.2 million/ cumm.

In most clinical hematology laboratories, an initial blood count is performed by an electronic instrument. Some of these instruments also produce a differential blood count, and a platelet count. Instruments that provide a 3-part differential indicate the percentage of neutrophils, lymphocytes, and a mixed field group that includes monocytes, eosinophils, basophils, immature and atypical cells. Thus, the atypical cells shown in the photograph would be counted as mixed cells and a smear review would be needed to make an identification. Instruments providing a 5-part differential count include monocytes and eosinophils. In cases where the mixed cell count is high, or there are other indications that atypical cells may be present, a hematologist's review of the smear is indicated.

The large cell illustrated in this photograph of a Wright/Giemsa-stained bone marrow smear is a megakaryocyte. This megakaryocyte appears mature. The nucleus has at least 8 lobes and the nuclear chromatin is coarse and distinct. Clusters of young platelets are being released from distinct platelet territories at the periphery of the cytoplasm (blue arrows). When mature, each megakaryocyte produces approximately 4000 platelets/day. Production can expand by 8-fold during times of increased demand and under the stimulus of thrombopoietin.

Several additional familial and congenital disorders associated with atypical inclusions in WBCs are now recorded. These individual syndromes carry the following names: Fechtner, Alport, Epstein, Sebastian, and Paris-Trousseau.Fechtner syndrome( Peterson etal,Blood 65:397-406,1985)was described with 8 family members spanning 4 generations presenting with varying degrees of nephritis, deafness,and congenital cataracts. The syndrome is likely a variant of Alport syndrome with the addition of leukocyte inclusions and macrocytothemia. Several more cases involving other families have been reported. The inclusions resemble toxic Doehle bodies or those of the May-Hegglin anomaly by light microscopy, but are ultrastructurally unique.Alport syndrome in itself is autosomal dominant, X-linked , hereditary and characterized by sensorineural deafness and hereditary nephritis. It is believed to result from abnormal glycopeptide synthesis in renal basement membranes. Recurrent hematuria and slowly progressive renal insufficiency are clinical findings. Cataracts and platelet abnormalities may be added features.Epstein syndrome is essentially Alport syndrome with the addition of macrothrombocytopenia (Seri, et al. Hum Genet 110:182-186, 2002). Neutrophil inclusions are absent in this disorder; neutrophilic inclusions are considered part of the Fechtner syndrome. The Sebastian platelet syndrome is a variant of hereditary macrothrombocytopenia combined with neutrophil inclusions that differ from Doehle bodies, but are similar to those inclusions in Fechtner syndrome. (Greinacher, et al, Blut 61:282-288, 1990).Paris-Trousseau syndrome includes large platelets containing giant alpha granules identifiable in the peripheral blood.(Breton-Gorius, Blood 85:1805,1995)

Illustrated in the upper photograph is a poorly defined cytoplasmic inclusion somewhat resembling a Doehle body. Note, however, that this inclusion is well defined and there is no evidence of toxic granulation in the cytoplasm.When Doehle-like bodies are identified, May-Hegglin anomaly should be considered in the differential diagnosis even though this entity is rare.The May-Hegglin anomaly is an inherited dominant condition in which large 2 - 5 um, basophilic and pyronophilic inclusions are present in granulocytes, including neutrophils, eosinophils, basophils, and monocytes.Similar to Doehle bodies, the May-Hegglin inclusions also are composed of RNA, probably derived from the rough endoplasmic reticulum. May-Hegglin anomaly includes giant platelets containing few fine granules (lower photograph).Sometimes the platelets have bizarre shapes and variable sizes. Variable degrees of thrombocytopenia complicated by mild bleeding problems and purpura may accompany the aberrant platelets.