Methods in hematology

Question 1

What is automated white blood cell differential count?

Answer 1

A differential blood count is a blood test to check your white blood cell levels, which can indicate the presence of infection, disease, or an allergic reaction.

Your doctor might order it as part of routine testing or to check for infections and other problems.

low expense
Highly automated

Question 2

What does an automated white blood cell differential count test used for?

Answer 2

• To enumerate the white blood cells and the major white blood cell types
• to enumerate the number of red blood cells by cell counting and haemoglobin analysis
• Determination of red blood cell indices:

= Average red blood cell size (MCV)
= Hemoglobin amount per red blood cell (MCH)
= The amount of hemoglobin relative to the size of the cell (hemoglobin concentration) per red blood cell (MCHC)

• To enumerate the number of platelets

Question 3

What are the steps involved in an automated white blood cell differential count?

Answer 3

• Use purple capped test tube with EDTA (dry EDTA crystals in tube)
• Whole blood containing RBC, WBC, and platelets in purple top tube.
• Cells flow in a column toward tubes with apertures through which blood can pass and are counted.

RBC lysed:

1) Hb measured from lysed RBCs
2) cells passing through the large aperture are WBCs - with flow cytometry in the circuit to identify WBC types by size and granularity.

RBCs not lysed:
1) all but a small percentage of cells passing through large aperture are RBCs, which are also sized as they pass through to determine their mean corpuscular volume (MCV)

• hematocrit or packed RBC volume is calculated from number and size of RBC.

Question 4

What is peripheral blood smear analysis?

Answer 4

A procedure in which a sample of blood is viewed under a microscope to count different circulating blood cells (red blood cells, white blood cells, platelets, etc.) and see whether the cells look normal.

low expense
smear prep automated or manual, followed by microscopic examination.

Question 5

What are the steps involved in a peripheral blood smear analysis?

Answer 5

• To determine the size, shape, and any abnormal morphology of all blood cell types by examining a stained preparation of blood cells microscopically.
• Sample collected in a purple capped test tube with EDTA.
• a drop of blood is applied to a glass slide and smeared to spread blood cells across the slide.
• microscopic examination is performed to detect abnormalities in number or in the appearance of:
• RBCs
• WBCs
• Platelets
• This test is commonly used early in the diagnostic process to assess a patient for an abnormality involving circulating blood cells.

Question 6

What is hemoglobin analysis?

Answer 6

A hemoglobin test measures the levels of hemoglobin in your blood. Hemoglobin is a protein in your red blood cells that carries oxygen from your lungs to the rest of your body. If your hemoglobin levels are abnormal, it may be a sign that you have a blood disorder.

low expense
semi-automated

Question 7

What is involved in a hemoglobin analysis?

Answer 7

• to determine the different Hb present by one or more methods that separate Hb types.
• Goal = identify the diff types of Hb present in a patient’s blood test

1) Blood collected in a purple test tube containing EDTA
2) RBC isolated by centrifugation and washing
3) RBC lysed and Hb released from cells
4) 2 methods
- Hb separated by electrophoresis and gel stained to reveal bands of Hb
- > Migrations of patient’s RBC compared to the migration of standard Hb (A, F, S, C ) on gel - using 1 or more electrophoresis systems.

or

• Hb types separated by isoelectric focusing (IEF) electrophoresis or by high-performance liquid chromatography (HPLC)

Question 8

What is sickle cell screening analysis?

Answer 8

• Test used to rapidly assess for the presence of Hb S by using methods involving either predisposition of red blood cells to sickle or the limited solubility of haemoglobin S.
• moderate expense
• manual assays, and the sickling test requires microscopic examination

Question 9

What is involved in a sickle cell screening assay?

Answer 9

• Sample of blood collected into purple top tube containing EDTA
• 2 available tests to detect sickle Hb
  = Sicking test and Solubility test
• Sickling test:
  1) blood onto glass slide
  2) Add reducing agent over droplet

-> Hb S detected by presence of Holly leaf or sickle cells upon microscopic exam.

• solubility test:
  1) blood added to a concentrated phosphate buffer solution
• followed by RBC lytic agent and reducing agent

-> Hb S detected if buffer becomes turbid because Hb S is not soluble in this buffer

Question 10

What diseases test positive for both the sickling and solubility test?

Answer 10

• Hb SS (sickle cell anaemia)
• Hb AS (sickle trait)
• Hb S with another Hb (example Hb SC)

Question 11

What are the erythrocyte sedimentation rate and C-reactive protein measurement?

Answer 11

• An erythrocyte sedimentation rate (ESR) is a type of blood test that measures how quickly erythrocytes (red blood cells) settle at the bottom of a test tube that contains a blood sample.

Normally, red blood cells settle relatively slowly.

A faster-than-normal rate may indicate inflammation in the body.

Inflammation is part of your immune response system.

It can be a reaction to an infection or injury.

Inflammation may also be a sign of a chronic disease, an immune disorder, or other medical condition.

low expense
manual or semiautomated

Question 12

What is the process of the erythrocyte sedimentation test?

Answer 12

goal: measure the height of sedimented RBC after an incubation, often 1 hour
- Whole blood placed in a cylindrical vessel with markings to assess column height
- RBC allowed to sediment undisturbed within cylindrical vessel.
- distance sedimented in mm/hr is erythrocyte sedimentation rate
- C-reactive protein, measured by an immunochemical method, is also used to assess systemic inflammation.

Question 13

What is produced inside bone marrow?

Answer 13

RBCs, WBCs, and platelets

bone marrow = soft fatty tissue inside bone cavities.

• Sometimes a sample of bone marrow must be examined to determine why blood cells are abnormal or why there are too few or too many of a specific kind of blood cell.

Question 14

What are the different types of bone marrow samples?

Answer 14

A doctor can take two different types of bone marrow samples:

Bone marrow aspirate: Removes fluid and cells by inserting a needle into the bone marrow and sucking out (aspirating) fluid and cells

Bone marrow core biopsy: Removes an intact piece of bone marrow using a coring device (similar to a large diameter needle)

Both types of samples are usually taken from the hipbone (iliac crest), often during a single procedure. Aspirates are rarely taken from the breastbone (sternum). In very young children, bone marrow samples are occasionally taken from one of the bones in the lower leg (tibia).

Question 15

What does a bone marrow aspirate show?

Answer 15

The bone marrow aspirate shows what cells, normal and abnormal, are present in the bone marrow and provides information about their size, volume, and other characteristics.

Special tests, such as cultures for bacteria, fungi, or viruses, chromosomal analysis, and analysis of cell surface proteins can be done on the sample.

Although the aspirate often provides enough information for a diagnosis to be made, the process of drawing the marrow into the syringe breaks up the fragile bone marrow. As a result, determining the original arrangement of the cells is difficult.

Question 16

What does a core bone marrow biopsy show?

Answer 16

The core biopsy removes an entire piece of bone marrow and shows not only what cells are present but also how full the bone marrow is with cells and where the cells are located within the marrow.

When the exact anatomic relationships of cells must be determined and the structure of the tissues evaluated, the doctor also does a core biopsy. A small core of intact bone marrow is removed with a special bone marrow biopsy needle and sliced into thin sections that are examined under a microscope.

Question 17

Where are bone marrow samples taken from?

Answer 17

Both types of samples are usually taken from the hipbone (iliac crest), often during a single procedure. Aspirates are rarely taken from the breastbone (sternum). In very young children, bone marrow samples are occasionally taken from one of the bones in the lower leg (tibia).

Question 18

How is bone marrow aspiration performed?

Answer 18

• The doctor or nurse makes a small incision in the skin, then inserts a hollow needle through the bone and into the bone marrow.
• Using a syringe attached to the needle, a sample of the liquid portion of the bone marrow is withdrawn.
• A bone marrow sampling begins with cleaning, sterilizing, and anesthetizing the skin over the bone. The procedure generally involves a slight jolt of pain, followed by minimal discomfort. The procedure takes a few minutes and causes no lasting damage to the bone.

Question 19

How is a bone marrow core biopsy performed?

Answer 19

• The provider will remove a small, solid piece of bone marrow using a special hollow needle. This is called a core biopsy. The biopsy needle will be removed. Firm pressure will be applied to the biopsy site for a few minutes, until the bleeding has stopped.
• A bone marrow sampling begins with cleaning, sterilizing, and anesthetizing the skin over the bone. The procedure generally involves a slight jolt of pain, followed by minimal discomfort. The procedure takes a few minutes and causes no lasting damage to the bone.

Question 20

What is anemia?

Answer 20

• Anemia is a decrease in the number of red blood cells (RBCs—as measured by the red cell count, the hematocrit, or the red cell hemoglobin content).

In men, anemia is defined as hemoglobin < 14 g/dL (140 g/L), hematocrit < 42% (< 0.42) , or RBC < 4.5 million/mcL (< 4.5 × 1012/L).

In women, hemoglobin < 12 g/dL (120 g/L), hematocrit < 37% (< 0.37), or RBC < 4 million/mcL (< 4 × 10 12/L) is considered anemia.

Question 21

Why should anemia be investigated?

Answer 21

• Anemia is not a diagnosis; it is a manifestation of an underlying disorder.
• Thus, even mild, asymptomatic anemia should be investigated so that the primary problem can be diagnosed and treated.

Question 22

What are the symptoms of anemia?

Answer 22

Anemia is usually suspected based on the history and physical examination.

Common symptoms and signs of anemia include:

• General fatigue
• Weakness
• Dyspnea on exertion
• Pallor
• The symptoms of anemia are neither sensitive nor specific and do not help differentiate between types of anemias.
• Symptoms reflect compensatory responses to tissue hypoxia and usually develop when the hemoglobin level falls well below the patient’s individual baseline.
• Symptoms such as weakness, fatigue, drowsiness, angina, syncope, and dyspnea on exertion can indicate anemia. Vertigo, headache, pulsatile tinnitus, amenorrhea, loss of libido, and gastrointestinal (GI) complaints may also occur. Heart failure or shock can develop in patients with severe tissue hypoxia or hypovolemia.
• Certain symptoms may suggest the cause of the anemia. For example, melena, epistaxis, hematochezia, hematemesis, or menorrhagia indicate bleeding. Jaundice and dark urine, in the absence of liver disease, suggest hemolysis. Weight loss may suggest cancer. Diffuse severe bone or chest pain may suggest sickle cell disease, and stocking-glove paresthesias may suggest vitamin B12 deficiency.

History and physical examination are followed by laboratory testing with a complete blood count and peripheral smear.

The differential diagnosis (and cause of anemia) can then be further refined based on the results of testing.

Question 23

What are the risk factors for anemia?

Answer 23

a vegan diet predisposes to vitamin B12 deficiency anemia,

alcoholism increases the risk of folate deficiency anemia.

A number of hemoglobinopathies are inherited, and certain drugs and infections predispose to hemolysis.

Cancer, rheumatic disorders, and chronic inflammatory disorders can suppress red cell production.

Question 24

What physical examinations can be done to diagnose anemia?

Answer 24

• A complete physical examination is necessary. Signs of anemia itself are neither sensitive nor specific; however, pallor is common with severe anemia.
• Signs of underlying disorders are more diagnostically accurate than are signs of anemia.
• Heme-positive stool identifies gastrointestinal bleeding.
• Hemorrhagic shock (eg, hypotension, tachycardia, pallor, tachypnea, diaphoresis, confusion) may result from acute bleeding.
• Jaundice may suggest hemolysis.
• Splenomegaly may occur with hemolysis, a hemoglobinopathy, connective tissue disease, myeloproliferative disorder, infection, or cancer.
• Peripheral neuropathy suggests vitamin B12 deficiency.
• Abdominal distention in a patient with blunt trauma suggests acute hemorrhage or splenic rupture.
• Petechiae develop in thrombocytopenia or platelet dysfunction.
• Fever and heart murmurs suggest infective endocarditis.
• Rarely, high-output heart failure develops as a compensatory response to anemia-induced tissue hypoxia.

Question 25

What type of testing can be performed to diagnose anemia?

Answer 25

• CBC with WBC and platelets
• RBC indices and morphology
• Reticulocyte count
• Peripheral smear
• Sometimes bone marrow aspiration and biopsy
• Laboratory evaluation begins with a complete blood count (CBC), including white blood cell (WBC) and platelet counts, RBC indices and morphology (mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC], red blood cell distribution width [RDW]), and examination of the peripheral smear.
• The reticulocyte count demonstrates how well the bone marrow is compensating for the anemia.
• Subsequent tests are selected on the basis of these results and on the clinical presentation.

Question 26

What is a complete blood count?

Answer 26

The automated CBC directly measures hemoglobin, RBC count, WBC count. and number of platelets, plus mean corpuscular volume (MCV, a measure of RBC volume). hematocrit (a measure of the percentage of blood made up of RBCs), mean corpuscular hemoglobin (MCH, a measure of the hemoglobin content in individual RBCs), and mean corpuscular hemoglobin concentration (a measure of the hemoglobin concentration in individual RBCs) are calculated values.

Question 27

What are microcytic and macrocytic RBCs?

Answer 27

• RBC populations are termed microcytic (small cells) if MCV is < 80 fL, and macrocytic (large cells) if MCV is > 100 fL.

However, because reticulocytes are also larger than mature red cells, large numbers of reticulocytes can elevate the MCV and not represent an alteration of RBC production.

Question 28

What do RBC indices indicate?

Answer 28

The RBC indices can help indicate the mechanism of anemia and narrow the number of possible causes.

Question 29

What is a Peripheral blood smear?

Answer 29

A procedure in which a sample of blood is viewed under a microscope to count different circulating blood cells

The peripheral smear is highly sensitive for excessive RBC production and hemolysis.

It is more accurate than automated technologies for recognition of altered RBC structure, thrombocytopenia, nucleated RBCs, or immature granulocytes and can detect other abnormalities (eg, malaria and other parasites, intracellular RBC or granulocyte inclusions) that can occur despite normal automated blood cell counts.

Question 30

What is a reticulocyte count?

Answer 30

• A reticulocyte count is a blood test that measures how fast red blood cells called reticulocytes are made by the bone marrow and released into the blood. Reticulocytes are in the blood for about 2 days before developing into mature red blood cells.

The reticulocyte count is expressed as the percentage of reticulocytes (normal range, 0.5 to 1.5%) or as the absolute reticulocyte count (normal range, 50,000 to 150,000/mcL, or 50 to 150 × 10 9/L).

The reticulocyte count is a crucial test in the evaluation of anemia because it informs about the response of the bone marrow and facilitates differentiation between deficient erythropoiesis (RBC production) and excessive hemolysis (RBC destruction ) as the cause of anemia.

• For example, higher values indicate excessive production (reticulocytosis); in the presence of anemia, reticulocytosis suggests excessive RBC destruction. Low numbers in the presence of anemia indicate decreased RBC production.