Hematology Lectures

Supportive Treatment of Multiple Myeloma

2016-07-18T01:36:00.001-07:00

Supportive Treatment of Multiple Myeloma

Supportive treatment is the treatment that aim to treat the signs & symptoms or reduce them; it is not aim to treat the disease itself.

Renal Impairment :-

Rehydrate.
Treat the underlying cause (e.g. hypercalcemia, hyperuricemia).
Dialysis.
Drink at least 3 Liter of fluid daily (all multiple myeloma cases).

Bone disease and hypercalcemia :-

Bisphosphonates (such as pamidronate, clodronate or zoledronic acid) reduce the progression of bone disease.
Rehydration with isotonic saline, a diuretic and corticosteroids followed by a biphosphonate (to treat acute hypercalcemia).

Compression paraplegia :-

Use decompression laminectomy or irradiation.
Also corticosteroid therapy may help.

Anemia :-

Erythropoietin.
Transfusion.

Bleeding (Bleeding caused by paraprotein interference with coagulation and hyperviscosity syndrome) :-

May be treated by repeated plasmapheresis.

Infections :-

Must treated rapidly.
Prophylactic infusions of immunoglobulin concentrates together with oral broad - spectrum antibiotics and antifungal agents may be needed for recurrent infections.

Multiple Myeloma: An Overview

2016-07-17T03:16:00.000-07:00

Multiple Myeloma: An Overview

Definition:-

Multiple myeloma (MM) is a malignant bone marrow–based, plasma cell neoplasm associated with abnormal protein production.

Multiple myeloma also called myelomatosis.

Epidemiology:-

1~2% of all types of malignant diseases
~10% of hematological malignancies.
15% of lymphoid malignancies.
~ 50 cases per million.
~ 50,000 Americans are diagnosed with MM each year.
Twice common in black than white people.
Slightly more common in malesthan in females.
98% of cases occur over the age of 40 years with a peak incidence in the 7th decade.

Etiology (Causes):-

- Exact cause is unknown, but the following are suspects:

Radiation.
Viral infections.
Toxins.
Chemicals.
Chromosomal abnormalities ( 8 % - 35 % of MM patients ).

- MM patients with chromosomal damage have a worse prognosis, a higher rate of disease acceleration, and decreased survival.

Clinical Signs & Symptoms:-

Fatigue: caused by anemia
Excessive thirst and urination: caused by excess calcium.
Nausea: caused by excess calcium.
Bone painin back and ribs: caused by plasma cell acceleration.
Bone fractures: caused by calcium leeching from bones into circulation.
Unexpected infections: caused by compromised immunity.
Weakness and numbness in the legs: caused by vertebrae compression.
Renal insufficiency.(Myeloma kidney).
Hypercalcemia.
Amyloidosis (in 5% of cases).
Weight loss & night sweats (in advanced cases).
Abnormal bleedingtendency: myeloma protein may interfere with platelet function and coagulation factors
Thrombocytopenia (in advanced cases).

Laboratory Findings:-

Anemia (2/3 of cases). ( Normocytic normochromic anemia ).
Leukopenia (1/3 of cases).
Thrombocytopenia.
ESR > 100 mm/hr.
Blood film shows rouleauxwith a bluish background staining.
Bone marrow shows >10% plasma cells.
↑ Total protein.
↑ Uric Acid.
↑ LDH.
Hypercalcemia.
Protein electrophoresis (monoclonal spike in the gamma region).
Bence-Jones protein (immunoglobulin light chain) in urine. (in some cases).
Serum β₂ microglobulin (β₂M) often raised and higher levels correlate with worse prognosis.

Radiology:-

X-rays, CT scan, MRI or PET.

Prognostic Data:-

Prognostic data include:-

Hb.
β₂M.
Creatinine.
Albumin. &
Extent of skeletal disease.

Treatment:-

Chemotherapy.
Supportive.
Radiation.
Transplantation (bone marrow; stem cell).

Prognosis:-

The prognosis of MM is poor.
Median survival of only 6 months without therapy.
The median survival can be increased to 3 years with chemotherapy.
Increased survival has been reported with autologous bone marrow & peripheral blood stem celltransplants.
Infection is a major cause of death.

Hemophilia: An Overview

2016-04-07T02:18:00.001-07:00

Hemophilia A (Classic Hemophilia; Factor VIII Deficiency)

Ø The most common inherited coagulation disorder (after Von Willebrand Disease).

Ø The factor VIII gene is on the X chromosome so inheritance is sex-linked with the severe disease occurring in males.

Ø Hemophilia A accounts for 80–85% of all cases of hemophilia with a prevalence of approximately 1 in 5,000–10,000 male births.

Ø Genetic changes of the factor VIII gene.

Ø Each son has a 50% chance of inheriting the affected gene.

Ø Approximately 30% of the affected individuals have no positive family history of the disease.

Clinical Features

Ø These range from severe spontaneous bleeding, especially into joints (hemarthroses) and muscles, to mild symptoms, depending on the factor VIII level.

Ø Generally individuals with > 30% activity do not have hemophilia symptoms.

Ø Onset in early childhood (e.g. post-circumcision).

Ø Pseudotumours as a result of extensive bleeds.

Ø Hemarthrosis (usually in severe cases) is the most common feature of severe hemophilia.

Ø Joint bleeds, particularly into the knee and ankle.

Ø Chronic debilitating joint disease caused by repeated bleeds.

Ø Increased risk of post-operative or post-traumatic hemorrhage.

Ø Subcutaneous hematomas can begin with slight trauma and spread to involve a large mass of tissue, causing purple discoloration of the skin.

Ø Epistaxis is rare in hemophilia.

Ø Hematuria.

Ø Deep muscle bleeding.

Ø Excess bleeding from dental extractions.

Ø Bleeding with intramuscular injections.

Ø Delayed bleeding after minor cuts.

Ø The most common cause of death (after exclusion of viral infections transmitted by the replacement product) is intracranial hemorrhage, which can occur spontaneously or after trauma.

Ø Mild deficiencies can be asymptomatic and unsuspected until a surgical procedure or major traumatic injury results in severe bleeding.

Laboratory Data

Ø APTT >> ↑

Ø PT >> N

Ø PFA-100 test >> N

Ø Plasma Factor VIII >> ↓

Ø Von Willebrand factor (vWF) >> N

Ø Carriers have factor VIII levels in plasma approximately 50% of normal. If the levels are <40% they may have clinical features of mild hemophilia. DNA analysis is helpful in carrier detection and antenatal diagnosis.

Treatment

Ø Infusions of factor VIII (either recombinant or concentrate from normal donated plasma “Cryoprecipitate”).

Ø Avoid aspirin, other antiplatelet drugs and intramuscular injections.

Ø Gene Therapy.

Complications of Treatment

Ø Infections.

Ø Neutralizing antibodies (inhibitors) to factor VIII in 15% of severe patients may require:

§ Immunosuppressive therapy,

§ Treatment with porcine factor VIII, or

§ Plasma exchange.

Hemophilia B (Factor IX Deficiency; Christmas Disease)

Ø 15–20% of hemophilia.

Ø ~1 in 30.000 males.

Ø Factor IX is coded by a gene close to the gene for factor VIII.

Ø Specific Factor IX Assay.

Ø Bleeding episodes are treated with high - purity factor IX concentrates. Because of its longer biological half-life, infusions do not have to be given as frequently as do factor VIII concentrates in haemophilia A.

Ø Recombinant factor IX.

Ø Gene Therapy.

Ø Clinical features & other laboratory data are same as in hemophilia A.

The Role of 2,3-Diphophoglycerate

2015-12-22T02:22:00.000-08:00

The Role of 2,3-Diphophoglycerate

The major function of the hemoglobin molecule is the transport of oxygen to the tissues. The oxygen affinity of the hemoglobin molecule is associated with the spatial rearrangement of the molecule and is regulated by the concentration of phosphates, particularly 2,3-DPG in the erythrocyte. The manner in which 2,3-DPG binding to reduced hemoglobin (deoxyhemoglobin) affects oxygen affinity is complex. Basically, 2,3-DPG combines with the beta chains of deoxyhemoglobin and diminishes the molecule’s affinity for oxygen.

When the individual heme groups unload oxygen in the tissues, the beta chains are pulled apart. This permits the entrance of 2,3-DPG and the establishment of salt bridges between the individual chains. These activities result in a progressively lower affinity of the molecule for oxygen. With oxygen uptake in the lungs, the salt bonds are sequentially broken; the beta chains are pulled together, expelling 2,3-DPG; and the affinity of the hemoglobin molecule for oxygen progressively increases.

In cases of tissue hypoxia, oxygen moves from hemoglobin into the tissues, and the amount of deoxyhemoglobin in the erythrocytes increases. This produces the binding of more 2,3-DPG, which further reduces the oxygen affinity of the hemoglobin molecule. If hypoxia persists, depletion of free 2,3-DPG leads to increased production of more 2,3-DPG and a persistently lowered affinity of the hemoglobin molecule for oxygen.

Figure: Hemoglobin molecular changes

Alterations in Myeloid:Erythroid Ratio

2015-12-11T11:04:00.001-08:00

The M:E ratio is sensitive to hematologic factors that may impair red blood cell life span, inhibit overall production, or cause dramatic increases in a particular cell line. Each of these conditions reflects bone marrow dynamics through alterations of the M:E ratio. Many observations in the peripheral smear can be traced back to the pathophysiologic events at the level of bone marrow. A perfect example of this is the response of the bone marrow to anemia. As anemia develops and becomes more severe, the patient becomes symptomatic, and the kidney senses hypoxia secondary to a decreased hemoglobin level. Tissue hypoxia stimulates an increased release of erythropoietin (EPO), a red blood cell-stimulating hormone, from the kidney. EPO travels through the circulation and binds with a receptor on the youngest of bone marrow precursor cells, the pronormoblast. Bone marrow has the capacity to expand production 6-8 times in response to an anemic event. Consequently, the bone marrow delivers reticulocytes and nucleated red blood cells to the peripheral circulation prematurely if the kidney senses hypoxic stress. What is observed in the peripheral blood smear is polychromasia (stress reticulocytes, large polychromatophilic red blood cells) and nucleated red blood cells. Both of these cell types indicate that the bone marrow is regenerating in response to an event, a dynamic that represents the harmony between bone marrow and peripheral circulation.

Bone Marrow & Myeloid:Erythroid Ratio

2015-12-11T10:20:00.001-08:00

The bone marrow is one of the largest organs of the body, encompassing 3% to 6% of body weight and weighing 1500 g in an adult. It is hard to conceptualize the bone marrow as an organ because it is not a solid organ that one can touch, measure, or weigh easily. Because bone marrow tissue is spread throughout the body, one can visualize it only in that context. It is
composed of yellow marrow, red marrow, and an intricate supply of nutrients and blood vessels. Within this structure are erythroid cells (red blood cells), myeloid cells (white blood cells), and megakaryocytes (platelets) in various stages of maturation, along with osteoclasts, stroma, and fatty tissue. Mature cells enter the peripheral circulation via the bone marrow sinuses, a central structure lined with endothelial cells that provide passage for mature cells from extravascular sites to the circulation. The cause and effect of hematologic disease are usually rooted in the bone marrow, the central factory for production of all adult hematopoietic cells. In the first 18 years of life, bone marrow is spread throughout all of the major bones of the skeleton, especially the long bones. As the body develops, the marrow is gradually replaced by fat until the prime locations for bone marrow in an adult become the iliac crest (located in the pelvic area) and the sternum (located in the chest area).
In terms of cellularity, there is a unique ratio in the bone marrow termed the myeloid:erythroid (M:E) ratio. This numerical designation provides an approximation of the myeloid elements in the marrow and their precursor cells and the erythroid elements in the marrow and their precursor cells. The normal ratio of 3:1 to 4:1 reflects the relationship between production and life span of the various cell types. White blood cells have a much shorter life span than red blood cells (6 to 10 hours for neutrophils as opposed to 120 days for erythrocytes) and must be produced at a much higher rate for normal hematopoiesis.

Iron Distribution

2015-12-10T18:21:00.000-08:00

Iron-containing compounds in the body are one of two types:

Functional compounds that serve in metabolic (hemoglobin, myoglobin, iron-responsive element-binding protein) or enzymatic (cytochromes, cytochrome oxygenase, catalase, peroxidase) functions
Compounds that serve as transport (transferrin, transferrin receptor) or storage forms (ferritin and hemosiderin) for iron.

A poorly understood iron compartment is the intracellular “labile pool.” Iron leaves the plasma and enters the intracellular fluid compartment for a brief time before it is incorporated into cellular components (heme or enzymes) or storage compounds. This labile pool is believed to be the chelatable iron pool. The total iron concentration in the body is 40–50 mg of iron/kg of body weight. Men have higher amounts than women.

Iron is found primarily in erythrocytes, macrophages, hepatocytes, and enterocytes (absorptive cells at the luminal [apical] surface of the duodenum). Hemoglobin constitutes the major fraction of body iron (functional iron) with a concentration of 1 gm iron/kg of erythrocytes, or about 1 mg iron/mL erythrocytes. Iron in hemoglobin remains in the erythrocyte until the cell is removed from the circulation. Hemoglobin released from the erythrocyte is then degraded in the macrophages of the spleen and liver, releasing iron. Approximately 85% of this iron from degraded hemoglobin is promptly recycled from the macrophage to the plasma where it is bound to the transport protein, transferrin, and delivered to developing normoblasts in the bone marrow for heme synthesis. The macrophages recycle 10 to 20 times more iron than is absorbed in the gut. This iron recycling provides most of the marrow’s daily iron requirement for erythropoiesis.

Iron in hepatocytes and intestinal enterocytes is stored and utilized as needed to maintain iron homeostasis. The hepatocytes store iron that can be released and utilized when the amount of iron in the plasma is not sufficient to support erythropoiesis. Enterocytes that absorb dietary iron can either export it to the plasma or store it. Iron stored in enterocytes is lost when the cells are sloughed into the intestine.

Erythrocyte

2015-12-07T08:47:00.001-08:00

Erythrocyte (Mature Erythrocyte)

Bone marrow : 0 %
Peripheral blood : Predominant cell type
Size : 7-8 μm
No nucleus
No nucleoli
No chromatin
No N/C ratio
Cytoplasm: Salmon with central pallor of about one-third of the diameter of the cell

NOTE: The mature erythrocyte has lost the blue-gray color and is salmon colored as hemoglobinization is complete.

Multiple Myeloma

2015-12-04T16:32:00.002-08:00

Multiple myeloma (myelomatosis) is a neoplastic disease characterized by plasma cell accumulation in the bone marrow, the presence of monoclonal protein in the serum and/or urine and, in symptomatic patients, related tissue damage.

Download Multiple Myeloma lecture (ppt)

Reticulocyte

2015-12-04T01:27:00.004-08:00

Reticulocyte ( Polychromatic Erythrocyte / Diffusely Basophilic Erythrocyte )

Size = 7-10 μm
No nucleus (so there is no N/C ratio)
No nucleoli
No chromatin
Cytoplasm: Color is slightly more blue/purple than the mature erythrocyte because there are RNA remnants.
Bone marrow: 1 %
Peripheral blood: 0.5 - 2.0 %

NOTE: When stained with supravital stain (e.g., new methylene blue), polychromatic erythrocytes appear as reticulocytes (contain precipitated ribosomal material)

Orthochromic Normoblast

2015-12-03T22:23:00.005-08:00

Orthochromic Normoblast ( Orthochromic Erythroblast / Metarubricyte )

1 - 4% of nucleated cells in BM
Peripheral blood: 0 %
Size = 10 -15 μm
Low N/C ratio (1:2)
Round nucleus
No nucleoli
Fully condensed chromatin
Cytoplasm: more pink or salmon than blue

NOTE: The gray-blue color of the cytoplasm is becoming salmon as more hemoglobin is produced.

Polychromatophilic Normoblast

2015-12-02T02:33:00.000-08:00

Polychromatophilic Normoblast ( Polychromatic Normoblast / Polychromatic Erythroblast / Rubricyte )

13-30% of nucleated cells in BM.
Peripheral Blood : 0 %
Size = 12 - 15 μm
Low N/C ratio (4:1)
Eccentric nucleus
No nucleoli
Chromatinirregular and coarsely clumped
Cytoplasm: Gray-blue as a result of hemoglobinization

NOTE: The blue color of the cytoplasm is becoming gray-blue as hemoglobin is produced.

Basophilic Normoblast

2015-12-01T23:36:00.001-08:00

Basophilic Normoblast (Basophilic Erythroblast / Prorubricyte)

1-3% of nucleated cells in BM.
Peripheral blood : 0 %
Size = 16-18 μm
Round to slightly oval nucleus
Moderate N/C ratio (6:1)
Dark blue cytoplasm
Indistinct nucleoli ( 0 - 1 nucleoli )
Coarsening ( slightly condensed ) chromatin

Pronormoblast

2015-12-01T09:34:00.001-08:00

Pronormoblast (also known as Proerythroblast / Rubriblast) is the first microscopically recognizable cell in erythrocyte lineage.

1% of Nucleated Cells in BM.
Peripheral Blood: 0%
1% of Nucleated Cells in BM.
Size: 20-25 μm
Nucleus: Round to slightly oval
High N/C ratio (8:1)
1-3 faint nucleoli
Fine chromatin
Cytoplasm: Dark blue; may have prominent Golgi

Glycosylated Hemoglobin

2015-12-01T08:20:00.001-08:00

HbA_1Con chromatography is a minor component of normal adult hemoglobin (HbA) that has been modified posttranslationally (HbA₃ on starch block electrophoresis). A component usually has been added to the N terminus of the β-chain. The most important subgroup of HbA₁is HbA_1C, which has glucose irreversibly attached. This hemoglobin is referred to as glycosylated hemoglobin. HbA_1Cis produced throughout the erythrocyte’s life, its synthesis dependent on the concentration of blood glucose. Older erythrocytes typically contain more HbA_1Cthan younger erythrocytes having been exposed to plasma glucose for a longer period of time. However, if young cells are exposed to extremely high concentrations of glucose ( >400 mg/dL) for several hours, the concentration of HbA_1Cincreases with both concentration and time of exposure.
Measurement of is routinely used as an indicator of control of blood glucose levels in diabetics because it is proportional to the average blood glucose level over the previous two to three months.

Average levels of HbA_1Care 7.5% in diabetics and 3.5% in normal individuals.

Structure of Hemoglobin

2015-12-01T06:44:00.003-08:00

Hemoglobin is the life-giving substance of every red blood cell, the oxygen-carrying component of the red blood cell. Each red blood cell is nothing more than a fluid-filled sac, with the fluid being hemoglobin. Every organ in the human body depends on oxygenation for growth and function, and this process is ultimately controlled by hemoglobin. In 4 months (120 days), red blood cells with normal hemoglobin content submit to the rigors of circulation. Red blood cells are stretched, twisted, pummeled, and squeezed as they make their way through the circulatory watershed.

The hemoglobin molecule consists of two primary structures:

1- Heme

This structure involves four iron atoms in the ferrous state (Fe²⁺ ), because iron in the ferric state (Fe³⁺ ) cannot bind oxygen, surrounded by protoporphyrin IX, or the porphyrin ring, a structure formed in the nucleated red blood cells. Protoporphyrin IX is the final product in the synthesis of the heme molecule. It results from the interaction of succinyl coenzyme A and delta-aminolevulinic acid in the mitochondria of the nucleated red blood cells. Several intermediate by-products are formed, including porphobilinogen, uroporphyrinogen, and coproporphyrin. When iron is incorporated, it combines with protoporphyrin to form the complete heme molecule. Defects in any of the intermediate products can impair hemoglobin function.
2- Globin

This structure consists of amino acids linked together to form a polypeptide chain, a bracelet of amino acids. The most predominant chains for adult hemoglobins are the alpha and beta chains. Alpha chains have 141 amino acids in a unique arrangement, and beta chains have 146 amino acids in a unique arrangement. The heme and globin portions of the hemoglobin molecule are linked together by chemical bonds.

2,3-Diphosphoglycerate (2,3-DPG)

2,3-DPG is a substance produced via the Embden-Meyerhof pathway during anaerobic glycolysis. This structure is intimately related to oxygen affinity of hemoglobin. As 2,3-DPG increased, the affinity of hemoglobin to oxygen is decreased.

Each hemoglobin molecule consists of four heme molecules with iron at the center and two pairs of globin chains. The heme structure sits lodged in the pocket of the globin chains. Hemoglobin begins to be synthesized at the polychromatic normoblast stage of red blood cell development. This synthesis is visualized by the change in cytoplasmic color from a deep blue to a lavender-tinged cytoplasmic color. Of hemoglobin, 65% is synthesized before the red blood cell nucleus is extruded, with an additional 35% synthesized by the reticulocyte stage. Normal mature red blood cells have a full complement of hemoglobin, which occupies a little less than one-half of the surface area of the red blood cell.

Figure: Hemoglobin molecule: note four heme molecules tucked inside globin chains.

Morphologic Characteristics of Erythroid Precursors

2015-11-28T17:07:00.000-08:00

Pronormoblast (rubriblast):

1% of Nucleated Cells in BM.
Size = 20-25 μm.
High N/C ratio (8:1)
1-3 faint nucleoli
Lacy chromatin

Basophilic normoblast (prorubricyte):

1-3% of nucleated cells in BM.
Size = 16-18 μm
Moderate N/C ratio (6:1)
Indistinct nucleoli
coarsening chromatin

Polychromatophilic normoblast (rubricyte):

13-30% of nucleated cells in BM.
Size = 12 - 15 μm
Low N/C ratio (4:1)
Chromatin irregular and coarsely clumped
Eccentric nucleus

Orthochromic normoblast (metarubricyte):

1-4% of nucleated cells in BM
Size = 10 -15 μm
Low N/C ratio (1:2)

Reticulocyte (new methylene blue stain):

Size = 7-10 μm
No N/C ratio because there is no nucleus

Reticulocyte (Wright’s stain):

Polychromatophilic (diffusely basophilic)

Mature RBC:

Size = 7 -8 μm
No nucleus

Thymus

2015-11-28T13:55:00.002-08:00

The thymus is a lymphopoietic organ located in the upper part of the anterior mediastinum. It is a bilobular organ demarcated into an outer cortex and central medulla. The cortex is densely packed with small lymphocytes (thymocytes), cortical epithelial cells, and a few macrophages. The medulla is less cellular and contains more mature thymocytes mixed with medullary epithelial cells, dendritic cells, and macrophages. The primary purpose of the thymus is to serve as a compartment for maturation of T lymphocytes. Precursor T cells leave the bone marrow and enter the thymus through arterioles in the cortex. As they travel through the cortex and the medulla, they interact with epithelial cells and dendritic cells, which provide signals to ensure that T cells can recognize foreign antigen but not self-antigen. They also undergo rapid proliferation. Only about 3% of the cells generated in the thymus exit the medulla as mature T cells. The rest die by apoptosis and are removed by thymic macrophages. The thymus is responsible for supplying the T-dependent areas of lymph nodes, spleen, and other peripheral lymphoid tissue with immunocompetent T lymphocytes.

The thymus is a well-developed organ at birth and continues to increase in size until puberty. After puberty, however, it begins to atrophy until in old age it becomes barely recognizable. This atrophy could be driven by increased steroid levels beginning in puberty and decreased growth factor levels in adults. The atrophied thymus is still capable of producing new T cells if the peripheral pool becomes depleted as occurs after the lymphoid irradiation that accompanies bone marrow transplantation.

FIGURE: A schematic drawing of the thymus. Hassall’s corpuscles are collections of epithelial cells that may be involved in the development of certain (regulatory) T cell subsets in the thymus.

Extramedullary Hematopoiesis

2015-11-28T13:29:00.001-08:00

Hematopoiesis in the bone marrow is called medullary hematopoiesis or intramedullary hematopoiesis.

Blood cell production in hematopoietic tissue other than bone marrow is called extramedullary hematopoiesis.

In certain hematologic disorders when hyperplasia of the marrow cannot meet the physiologic blood needs of the tissues, extramedullary hematopoiesis can occur in the hematopoietic organs that were active in the fetus, principally the liver and spleen. Organomegaly frequently accompanies significant hematopoietic activity at these sites. This extramedullary hematopoiesis in postnatal life reflects the ability of inert hematopoietic cells to become active, functional cells if the need arises.

Sites of Hematopoiesis

2015-11-26T13:39:00.000-08:00

Hematopoiesis/Hemopoiesis is the process by whitch blood cells are formed.

Sites of Hematopoiesis

Yolk sac

From the 18^thday after fertilization, the yolk sac begins hematopoiesis. The cells made here are erythrocytes & few macrophages.

Aorta-gonads-mesonephros (AGM) region

Located along the developing aorta. This region has the ability to make a wider range of hematopoietic cells including lymphocytes.

At about the 3^rd month of fetal life, the yolk sac & AGM discontinue their role in hematopoiesis.

Liver

At about the 3^rd month of fetal life, the liver becomes the chief site of blood cell production. The liver continues to produce a high proportion of erythroid cells, but myeloid and lymphoid cells begin to appear in greater numbers.

As fetal development progresses, hematopoiesis also begins to a lesser degree in the spleen, kidney, thymus, and lymph nodes. Erythroid and myeloid cell production as well as early B cell (lymphocyte) development gradually shifts from these sites to bone marrow during late fetal and neonatal life as the hollow cavities within the bones begin to form.

Bone Marrow

The bone marrow becomes the primary site of hematopoiesis at about the 6^th month of gestation and continues as the primary source of blood production after birth and throughout adult life.

The thymus becomes the major site of T cell (lymphocyte) production during fetal development and continues to be active throughout the neonatal period and childhood until puberty.

Lymph nodes and spleen continue as an important site of late B cell differentiation throughout life.

NOTE

Liver & spleen may return to hematopoiesis after birth if necessary in a process called extramedullary hematopoiesis (production of blood cells outside the bone marrow).

Activated Partial Thromboplastin Time (APTT / aPTT / PTT) Test Procedure

2015-11-24T14:10:00.000-08:00

Principle

Platelet-poor plasma is added to an equal volume of partial thromboplastin reagent and warmed to 37°C for an exact incubation time. Pre-warmed (37°C) calcium chloride reagent (0.025M) is added to this mixture to activate the coagulation cascade. The time required for clot formation is recorded. Clot formation may be detected by optical or electromechanical methods using manual, semi-automated, or automated devices.

Reagents and Equipment

Partial thromboplastin reagent: consists of phospholipids and a contact activator
Calcium chloride reagent, 0.025M
Fibrometer system
a. Fibrometer
b. Thermal prep block, 37 + 1°C
c. Automatic pipet
Coagulation cups, non-wettable surface
Fibro-tips

Quality Control

Quality control materials (normal and abnormal) with established control limits should be run. NCCLS recommends controls be tested at the beginning of each testing day, followed by testing during each subsequent shift or with each batch of assays.

Specimen

Whole blood anticoagulated with 3.2% sodium citrate is the specimen of choice. The specimen should be processed to obtain platelet-poor plasma.

Procedure (Fibrometer Testing)

Pre-warm a sufficient quantity of calcium chloride reagent to 37°C for the number of tests to be performed.
Pipet 0.1 mL patient sample or control sample into labeled coagulation cup using the automatic pipet.
To each sample cup, add 0.1 mL of partial thromboplastin reagent using the automatic pipet. Carefully mix the contents of each cup.
Allow sample-partial thromboplastin reagent mixture to pre-warm to 37°C for 1-2 minutes. No longer than five minutes.
Pipet 0.1 mL pre-warmed calcium chloride reagent into coagulation cup containing sample-partial thromboplastin reagent mixture using the automatic pipet (switch in the "on" position). When the calcium chloride reagent is dispensed the timer will automatically start. Alternatively, the timer may be started by touching the timer plate. Reagent should be forcibly added to ensure mixing of reagent and sample.
The timer will stop when clot formation occurs.
Record time taken for clot formation.
Each sample (patient or control) should be run in duplicate.

Results

The average of the duplicate partial thromboplastin times are recorded to the nearest second.

Reference Interval

Each laboratory should establish its own reference interval following a recommended procedure. The reference interval should be re-established with changes in instrumentation, reagent lot number, or at least once a year.

Comments

1- Partial thromboplastin reagent consists of phospholipids and a contact activator. Kaolin, celite, silica, and ellagic acid are examples of activators available. Care should be taken in choosing the partial thromboplastin reagent, since reagents vary in their sensitivity or insensitivity to lupus anticoagulant.

2- Each laboratory should determine the optimal incubation time for partial thromboplastin reagent and sample based on its assay procedure. The longer the incubation time, the shorter the partial thromboplastin times due to increased contact activation. Excessive heating (>5 minutes) will result in loss of Factor V and Factor VIII.

3- Precision between duplicate measurements is said to be acceptable if the difference between duplicates is 10% or less of the mean of the duplicates.

4- The APTT has been used to monitor heparin therapy. However, newer methodologies such as Factor Xa inhibition assay are replacing this use of the APTT.

5- The APTT is prolonged in:

Inherited single factor deficiencies of factors XI, X, IX, VIII, V, II, and I.
Disseminated intravascular coagulation (DIC).
Presence of circulating inhibitors like lupus-like anticoagulant.

6- If the patient's hematocrit exceeds 55%, NCCLS recommends adjusting the amount of anticoagulant used in the collection tube to prevent over-anticoagulation of the specimen. This correction formula is:

C = (1.85 x 10^-3) X (100 - Hct) X V

Where: C = volume of sodium citrate, V = volume of whole blood drawn, Hct = patient's hematocrit.

Note

Potential sources of error

Associated with specimeninappropriate ratio of anticoagulant to blood
failure to correct citrate volume if hematocrit >55%
clotted, hemolyzed, icteric, or lipemic specimen
delay in processing or testing
inappropriate storage
Associated with reagentsincorrect preparation of reagents
failure to properly store reagents
use of reagents beyond reconstituted stability time
use of reagents beyond expiration date
contaminated reagents
Associated with procedure
incorrect temperature
incorrect incubation times
incorrect volumes of sample, reagents, or both
improperly functioning instrument

Thrombin Time (TT) Test

2015-11-23T13:21:00.000-08:00

TT has an important role as a screening test because it measures the conversion of fibrinogen to fibrin by adding excess thrombin to undiluted plasma. Because the additional clotting factors previously measured in the PT and APTT have no effect on this test, TT is generally useful for evaluating other parameters affecting the formation of fibrin. There can be interference with the conversion of fibrinogen to fibrin for three major reasons: the presence of hypofibrinogenemia or dysfibrinogenemia, the presence of heparin, and the presence of fibrin degradation products (FDP). In rare cases, autoantibodies against thrombin (e.g., induced by topical thrombin application or the use of fibrin sealants) and myeloma proteins can also interfere with fibrin formation and result in an abnormal TT. The TT is useful in corroborating an abnormal FDP result and can verify that the citrated blood sample was drawn through an indwelling heparinized catheter that was not well flushed. An extremely prolonged TT usually indicates a heparin effect. If the sample is contaminated with heparin, it can be absorbed with Hepzyme. The testing can then be repeated, or the specimen can be redrawn.

The general reference interval for the TT is 10–16 seconds. TT’s sensitivity can be increased by diluting the thrombin reagent to give a control of 16–18 seconds. The TT in preterm and term infants is longer than the adult reference interval even though the fibrinogen level is within the same normal reference interval, which can be explained by the presence of a distinct fetal fibrinogen molecule with altered function. The TT generally becomes normal within a few days after birth.

Thrombin time procedure

Pre-warm 0.1 mL patient or control plasma.
Add 0.2 mL pre-warmed thrombin reagent and start timing device.
Stop timing device upon formation of a clot.

Note: Optimal reaction temperature is 37°C.
Reference interval: 10-16 seconds.

NOTE: Conditions associated with prolonged thrombin time

Hypofibrinogenemia

Dysfibrinogenemia

Paraproteins (e.g., cryoglobulin)

Presence of heparin

Presence of fibrin degradation products

Presence of plasmin

Qualitative D-Dimer Test

2015-11-23T05:25:00.000-08:00

Principle

D-dimer is a fibrin fragment that results when plasmin acts on cross-linked fibrin in the presence of factor XIII. D-dimers are produced from an insoluble fibrin clot. Available since the 1990s, this semiquantitative assay provides evidence of normal or abnormal levels of D-dimer. Latex particles are coated with mouse anti–D-dimer monoclonal antibodies. When mixed with plasma containing D-dimers, agglutination occurs. The test plays an important role in detecting and monitoring patients suspected to have thrombotic disorders. Its clinical uses include:

Detecting deep vein thrombosis (DVT)
Pulmonary embolism (PE)
Disseminated intravascular coagulation (DIC)
Postoperative complications
Septicemia.

Quantitative D-dimer procedures are available, using latex-enhanced turbidimetric methods. The qualitative test is widely used in most coagulation laboratories, however, as a screening test for D-dimers.

Reagents and Equipment

1. D-dimer kit containing reagents (stored at 2 to 8°C), good until expiration date on the kit
    a. Test reagent solution containing red blood cell anti–XL-FDP antibody conjugate
    b. Negative control solution containing 0.9% saline solution
    c. Positive control solution containing purified D-dimer fragment
2. Plastic agglutination trays

3. White plastic stirrers
4. Timer
5. 10-μL pipette with disposable tips

Specimen Collection and Storage

1. Collect venous whole blood into a vacuum tube with 3.2% sodium citrate. Dilution of blood to anticoagulant must be 9:1 .
a. 4.5 mL of blood with 0.5 mL of anticoagulant, or
b. 2.7 mL of blood with 0.3 mL of anticoagulant
2. Collection of venous blood into heparin is acceptable.
3. Store specimens at 18 to 24°C. Specimens should be tested within 4 hours of specimen collection. If testing will take place after 4 hours, specimens must be refrigerated at 2 to 8°C; refrigerated specimens are good up to 24 hours.

Quality Control

1. Quality control is performed under several conditions:
    a. Daily
    b. When opening a new kit
    c. When receiving a new shipment
    d. When a new lot number is put into use
2. A whole blood sample that has a negative D-dimer result is used for quality control.
3. Quality control method
    a. Follow directions in the procedure to do the quality control, steps 1 through 5.
    b. Add 1 drop of positive control to the test well, and proceed with steps 6 through 8b.

Procedure

1. Allow reagents to come to room temperature for at least 20 minutes before use.
2. Specimen should be thoroughly mixed; do not allow cells to settle out.
3. For each sample, pipette 10 μL of whole blood into each reaction well; place wells on a plastic agglutination tray; the first is labeled “negative control well” and the second is labeled “test well.”
4. Add 1 drop of the negative control to the negative control well.
5. Add 1 drop of the test reagent to the test well.

6. With a plastic stirrer, mix the contents of each well thoroughly for 3 to 5 seconds, using a different stirrer for each well and spreading the reagent across the entire well surface.
7. To promote agglutination, mix by gentle rocking of the plastic agglutination tray for 2 minutes.
8. At the end of the 2 minutes, observe for the presence of agglutination.
    a. Positive results: Agglutination is present in the test well compared with no agglutination
in the negative control well.
    b. If the negative control well agglutinates, the test is invalid.
    c. If the test result is negative, add 1 drop of positive control to the test well and rock the plastic tray. Agglutination should occur within 15 seconds. If agglutination does not occur with the addition of the positive control, the test is invalid.

Interpretation

1. Positive: Agglutination seen in the test well and no agglutination seen in the negative control well.
2. Negative: No agglutination seen in the test well and the negative control well. This would be confirmed by adding the positive control to the test well and observing agglutination.
3. Invalid
a. Agglutination occurs in the negative control well.
b. No agglutination occurs with the positive control.

Results

Negative: No agglutination seen in negative agglutination well (less than 0.5 mg/L)
Positive: Agglutination seen in undiluted sample (0.5 to 4.0 mg/L)
Positive samples can be diluted 1:8 or 1:64 to provide more specific semiquantitative data on the amount of D-dimer present.

Limitations

The presence of cold agglutinins in patient samples can cause agglutination in patient blood. This may cause agglutination of the negative control, invalidating the test results.
A quantitative D-dimer test is available, and it is usually performed using a turbidimetric procedure on automated coagulation equipment. The advantage of this method is that it gives an absolute quantity of D-dimer in milligrams per liter, which is an effective tool for determining whether a thrombotic episode has occurred or predicting whether one will occur.

Prothrombin Time (PT) Manual Test Procedure

2015-11-22T18:47:00.000-08:00

Principle

The addition of pre-warmed (37°C) platelet-poor plasma to thromboplastin-calcium reagent activates the coagulation cascade at Factor VII. The time required for clot formation is recorded. Clot formation may be detected by optical or electromechanical methods using manual, semi-automated, or automated devices.

Reagents and Equipment

Thromboplastin-calcium reagent
Fibrometer system
a. Fibrometer
b. Thermal prep block, 37 + 1°C
c. Automatic pipet
Coagulation cups, non-wettable surface
Fibro-tips

Quality Control

Quality control materials (normal and abnormal) with established control limits should be run. NCCLS recommends controls be tested at the beginning of each testing day, followed by testing during each subsequent shift or with each batch of assays.

Specimen

Whole blood anticoagulated with 3.2% sodium citrate is the specimen of choice. The specimen should be processed to obtain platelet-poor plasma.

Procedure (Fibrometer Testing)

Pre-warm a sufficient quantity of thromboplastin-calcium reagent to 37°C for the number of tests to be performed.
Pipet 0.1 mL patient sample or control sample into labeled coagulation cup using the automatic pipet.
Allow sample to pre-warm to 37°C for 1-2 minutes. No longer than five minutes.
Pipet 0.2 mL pre-warmed thromboplastin-calcium reagent into coagulation cup containing patient or control sample using the automatic pipet (switch in the "on" position). When the thromboplastin-calcium reagent is dispensed the timer will automatically start. Alternatively, the timer may be started by touching the timer plate. Reagent should be forcibly added to ensure mixing of reagent and sample.
The timer will stop when clot formation occurs.
Record time taken for clot formation.
Each sample (patient or control) should be run in duplicate.

Results
The average of the duplicate prothrombin times is recorded to the nearest half -second. To report results in INR, use the following calculation:

INR = (patient's PT/mean PT of the normal range)^ISI

Reference Interval

Each laboratory should establish its own reference interval following a recommended procedure. The reference interval should be re-established with changes in instrumentation, reagent lot number, or at least once a year.

Comments

Precision between duplicate measurements is said to be acceptable if the difference between duplicates is 10% or less of the mean of the duplicate results.
If the patient's hematocrit exceeds 55%, NCCLS recommends adjusting the amount of anticoagulant used in the collection tube to prevent over-anticoagulation of the specimen. This correction formula is:

C = (1.85 x 10^-3) X (100 - Hct) X V

Where: C = volume of sodium citrate, V = volume of whole blood drawn, Hct = patient's hematocrit.
Significant variation in PT results is observed among different commercial thromboplastin-calcium reagents due to the source of thromboplastin. To normalize these variations, each thromboplastin-calcium reagent has an established international sensitivity index (ISI) that is determined by comparison to the gold standard thromboplastin used by the World Health Organization.The reagent's ISI is used in the calculation of the international normalized ratio (INR). Failure to use the ISI established for a particular lot number of thromboplastin-calcium reagent will result in errors in the INR result. Since the INR is used to monitor patients on oral anticoagulant therapy, an error could lead to misinterpretation of the patient's anticoagulation status.
The prothrombin time is prolonged in individuals with inherited single factor deficiencies of factors X, VII, V, II, I; individuals with liver disease; individuals with vitamin K deficiency; individuals with disseminated intravascular coagulation; and individuals taking oral anticoagulants.

NOTE:

Potential sources of error:-
1. Associated with specimen:-Inappropriate ratio of anticoagulant to blood.
  Failure to correct citrate volume if hematocrit >55% .
  Clotted, hemolyzed, icteric, or lipemic specimen.
  Delay in processing or testing.
  Inappropriate storage.
2. Associated with reagents:-Incorrect preparation of reagents.
  Failure to properly store reagents.
  Use of reagents beyond reconstituted stability time.
  Use of reagents beyond expiration date.
  Contaminated reagents.
3. Associated with procedure:-Incorrect temperature.
  Incorrect incubation times.
  Incorrect volumes of sample, reagents, or both.
  Improperly functioning instrument.

Hemacytometer

2015-11-22T13:42:00.003-08:00

Cell counts are performed manually by diluting blood with a diluent, loading a small amount of the diluted sample on a ruled device (hemacytometer), and counting the cells microscopically. The hemacytometer consists of two side-by-side identically ruled glass platforms mounted in a glass holder. Each platform contains a ruled square measuring 3 X 3 mm (9 mm²) and is subdivided according to the improved Neubauer ruling. This ruling subdivides the ruled square into 9 large squares, each measuring 1 X 1 mm (1mm²). All 9 squares are used for leukocyte counts. The large center square is used for platelet and erythrocyte counts. This center square is divided into 25 smaller squares, each with an area of 0.04 mm². The 5 squares labeled R are used in performing the erythrocyte count whereas the entire center square is used in performing the platelet count.

On either side of the two ruled glass platforms is a raised ridge. The coverglass is placed on top of the ridge. The distance between the coverglass and the surface of the ruled
area (depth) is exactly 0.1 mm. Thus, the ruled area on each side of the hemacytometer holds a volume of 0.9 mm³(3 X 3 X 0.1).

The unopette system is a fast and accurate method for collecting and diluting blood for cell counts. For each laboratory determination performed, the unopette consists of the following elements: the reservoir, the pipet, and the pipet shield. The reservoir contains a premeasured volume of diluting fluid that is specific for the cell count to be performed.

Figure: Unopette system (from left to right): pipet shield, pipet, and reservoir.

In summary:

Figure: Neubauer hemacytometer counting area. The entire counting area is 9 mm²(3 mm × 3 mm) and is divided into 9 squares. Each square is 1 mm²(1 mm × 1 mm) in area. Using the 10× objective, 1 square of the counting area (1 mm²) can be viewed. For the manual leukocyte count, all 9 squares are counted. The center square is further divided and the R squares are used for erythrocyte counts, while the entire center square is used for platelet counts.