Special Hematologic Examination

8/4/2019 Special Hematologic Examination

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Review: STEM CELL LINEAGE AND DIFFERENTIATION

(kindly check your histology books for this one)

SUMMARY ON BLOOD COAGULATION

Function of the Coagulation System

1. Controlling bleeding2. To maintain blood in a clot free state (thrombosis

should be controlled)

3. To maintain the integrity of the endothelium due toendothelial injury

Key Regulators of Coagulation System (NEVERFORGET!):

1. VASCULAR WALL2. COAGULATION SYSTEM3. PLATELETS

Platelet defects can be classified as:

a. Quantitative : either there is a decrease orincrease in the number of platelets.

- ThrombocytoPENIA = decrease inplatelet count

- ThrombocyTOSIS = increase in plateletcount

- REMEMBER: normal platelet count =150,000-450,000/L of blood

(labtestsonline.com)

b. Qualitative : defect is on the surface membraneprotein or granules (factors that contribute to

platelet integrity and function)

Examples of Qualitative Disorders

Glanzmanns thrombasteniaDefect :

- Failure of the platelets to aggregate inresponse to a normal stimuli

- Absence or reduced platelet glycoprotein(GP) IIb or IIIa as well as fibrinogen

receptors needed for aggregation

Characteristics:

- Inherited, autosomal recessive- Platelets appear normal, platelet count is

normal and platelets adhere to exposed

subendothelial proteins.

Prolonged BT but Normal PT & APTT

BT = Bleeding time

PT = Prothrombin time

APTT = Activated partial thromboplastin time

Bernard Soulier SyndromeDefect :

- Deficiency of platelet surfaceglycoproteins (GP) Ib / Ix

Characteristic:

- Autosomal recessive disorderProlonged BT, large platelets,

thrombocytopenia

- Bleeding occurs because affectedplatelets do not adhere to the Von

Willebrand factor in the subendothelial

matrix

There are several platelet function disorders

relating to platelet secretion, which refers to

the release of the contents of platelet

granules that occurs following platelet

activation

- This includes:Gray Platelet syndrome

Chediak-Higashi syndrome (CHS)

Dense Granule deficiency

Review questions:

What is the common congenital bleeding disorder?

Ans : Von Willebrand Disease

What is the common congenital coagulation disorder?

Ans : Factor VIII or Hemophilia A

What test is used to detect coumarin or heparin?

Ans: PT (Prothrombin Time)

Test to detect Heparin? PTT

Test for Intrinsic factor? PTT (APTT)

Test for extrinsic factor? PT

Lecture Proper

Special Hematologic Test

Categories:

I. Tests to determine presence of hemolysis(hemolytic anemia)

II. Hemoglobin electrophoresisIII. Bone marrow examination (bone marrow

aspirate and biopsy)

Subject: Pathology (CP)

Topic: Special hematologic tests

Lecturer: Dr. Pascual

Date of Lecture: August 18, 2011

Transcriptionist/Editor: RAAJAH

Pages:SY

2011-2012


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I. TESTS TO DETERMINE PRESENCE OF HEMOLYSIS(HEMOLYTIC ANEMIA)

A.RETICULOCYTE COUNT- A measure of marrow erythropoiesis (bone

marrow production of RBCs)

Principle:

- Reticulocytes are immature, non-nucleatedRBCs with remnants of RNA.

- Stage between orthochromatic normoblast andmature erythrocytes

Procedure:

1. Mix an equal amount of patients blood witha supravital stain

** supravital stain may be either Methylene

blue or brilliant cresyl blue

** cytoplasmic RNA does not take up the

stain used normally for CBC that is why

supravital stain is needed.

**Supravital stain - test used for detecting

reticulocytes

**Giemsa or wright stain - used for CBC

2. Incubate for 15 minutes3. Make a smear4. Count the number of reticulocytes that you

see in 1000 RBC/10

Normal value:

Reticulocyte : 0.005 0.015 or (0.5 1.5%)

**divide the # of reticulocytes by 10 if you want

to get the percent equivalent.

Interpretation:

- An increase in reticulocyte count indicates anincrease in bone marrow erythropoiesis

- A decrease in reticulocyte count indicates adecrease in bone marrow erythropoiesis

Indications:

- May be used in the diagnosis of hemolyticanemia

**Remember: in hemolytic anemia there is no defect

in RBC production but rather, this may reflect an

increased RBC destruction in the circulation

**Hemolytic anemia = increased reticulocyte count

due to the increased destruction in the periphery

(circulation), the bone marrow tries to compensate

by producing more red cell precursors (reticulocytes)

- May be used to monitor patients response toiron therapy in the treatment of iron deficiencyanemia

B.DIRECT ANTIGLOBULIN (COOMBS TEST)Principle:

- Some forms of hemolytic anemia can be due toiso-antibodies or auto-antibodies which detects

red cell membrane antigens as foreign

o These antibodies then attach to the cellmembrane antigen and induce hemolysis

- Direct Coombs test detects the coating of thered cell by these antibodies (iso-antibodies and

auto-antibodies attached to red cells)

Direct coombs test detects in-vivo coating of RBCs by

antibodies

Px RBC (coated w/ Ab) + Coombs reagent (AntiH & Ab)

Procedure

- Patients RBCs (coated with antibody) are mixedwith commercially prepared Coombs reagent

(antihuman globulin antibody)

**If patient red cells are coated by antibodies, anti-

human antibodies (reagent) will bind to the patient

antibodies = AGGLUTINATION

Interpretation:

(+)result : + Agglutination = POSITIVE Coombs Test

(-) result : - Agglutination = NEGATIVE Coombs TestIndications:

- May be used in the diagnosis of the following:o hemolytic disease of the newborno hemolytic anemia in adultso hemolytic transfusion reactions

** In all these settings, you have antibodies attached to

the red cell membrane

C. OSMOTIC FRAGILITY TEST / INCUBATED OSMOTICFRAGILITY TEST

- Measures the ability of the red cells to take upfluid without lysing

- Fragility of the cell is primarily dependent on theshape of the cell

- Assesses primary factors that determine how redblood cells react in the osmotic fragility test

- Requested if one is suspecting congenitalhereditary spherocytosis

3 primary factors that determine how red blood cellsreact in the osmotic fragility test

1. Functional status of the cell membrane2. Volume of the cell3. Surface area of the cell

Best example is a spherocyte - due to its smaller

surface area resulting from a defect in its red cell

cytoskeleton, it is more fragile

Principle:

- Red cells placed in an isotonic solution, usually a0.85 0.9 NaCl solution, the fluid will neitherenter or leave the cell

- Placing the red cells in a hypotonic solution, (eg.0.25 NaCl solution), the cells swell and rupture.

- Spherocytes (defect with ankerin) are moresusceptible to hemolysis in hypotonic solution

than normal RBC that hemolyse at

concentrations above normal


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Procedure:

1. Mix patients blood with NaCl solution ofdecreasing concentration

2. Centrifuge and incubate for 30 mins at roomtemperature (osmotic fragility test)

** incubate it for 24 hours for incubated

osmotic fragility test

3. Examine the optical density of the supernatantusing a spectrophotometer4. Calculate for % hemolysis using the formula:

% Hemolysis = OD of the supernatant x 100

OD of the test tube (no NaCl)

**OD Optical Density

Reference values

Hemolysis usually begins at 0.50 NaCl concentration and

is completed at 0.30 NaCl concentration

**increased osmotic fragility hemolysis starts at a

higher concentration (0.55 1 NaCl)

**decreased osmotic fragility hemolysis starts at

lower concentration less than 0.30 NaCl (normal)

NORMAL VALUES

NaCl

Concentration

%

Hemolysis

1.00 0.55 0

0.50 0 5

0.45 0 45

0.40 50 90

0.35 90 99

0.30 97 100

0.20 0.10 100

Interpretation:

- INCREASED OSMOTIC FRAGILITY is usuallyobserved in cases of congenital spherocytosis

and acquired hemolytic anemia

- DECREASED OSMOTIC FRAGILITY (DOF) is usuallyobserved in cases of iron deficiency anemia,

thalassemia and sickle cell anemia** disease entities of DOF shows target cells in the

periphery as seen in thalassemia, severe iron

deficiency anemia and some liver diseases.

Indications:

- May be used in the diagnosis of CongenitalSpherocytosis

o This inherited disorder is caused by intrinsicdefects in the red cell membrane

Abnormally in the red cell membrane

cytoskeleton (proteins)

Weakness of themembrane

o Morphology: the spherocytosis appear asmicrocytes without central pallor:

abnormally small, dark staining

(hyperchromic) red cells lacking the normal

central zone of pallor

o Moderate splenic enlargement ischaracteristic (500 gm to 1000 gm). It results

in the congestion of the cords of Billroth and

work hyperplasia due to markedly

increased erythrophagocytosis

o When these red cells go in the circulationbecause of this membrane instability, there

would be fragmentation of red cellmembrane in order for the red cell to

compensate and contain the same amount

of hemoglobin, it will assume a spherical

shape less deformable less elasticwhen

it is in the spleen it will not be able to leave

the spleen making it stay longer in the spleen

and be more exposed to phagocytic cells

hemolysis chronic hemolytic anemia

D. ACID SERUM (HAMS) TEST-

This test is used in the diagnosis ofparoxysmalnocturnal hemoglobinuria

Principle:

- Patients with PNH (paroxysmal nocturnalhemoglobinuria) are more susceptible to

complement mediated hemolysis because of a

red cell membrane defect

** in PNH you also have hemolytic anemia

**basic defect in PNH can be traced to the cell

membrane which is susceptible to complement

mediated hemolysis due to a defect in GP-I

(glycoprotein-1) membrane enzymes.

** CD 55 or decay accelerating factor

** CD 59 or membrane inhibitor of reactive lysis

** CD 8

Procedure:

1. Patients red cells are mixed with normal serum(ABO compatible serum), with the patients own

serum and with normal serum inactivated to

destroy complement.

2. Patients washed RBC + ABO compatible normalserum + a weak acid (0.15 NHCL)

** when you acidify the mixture, you are

actually activating complement by the

alternative pathway and facilitates binding of C3

to the RBC membrane

** Control: using normal red cells

3. Incubate at 37C for 1 hour** normal acidified serum and treated serum

prepared by incubation at 56C for 30 minutes to

inactivate complement activity are also added to

the patient and control cells in separate tubes.

4. Determine the percent hemolysed red cells.5. (+) result: >10-15% RBC hemolysed

II. HEMOGLOBIN ELECTROPHORESIS- Used to detect abnormal hemoglobin

*Ex. Hemoglobin S Sickle Cell Anemia

Hemoglobin H Thalassemia


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Hemoglobin A Normal

- Used to diagnose hemoglobinopathyPrinciple:

- Hb molecules in alkaline solution have a net (-)charge and migrate toward the anode in an

electrophotometric system

Procedure

1. Patients hemolysate is placed in a celluloseacetate membrane and immersed on both endsin buffered solution.

** a hemolysate is prepared from the patients

blood sample and placed on the cathode side of

the acetate strip because they ave a net negative

charge in an alkaline solution so they will migrate

toward positive side.

2. Electric current is applied and allows Hb tomigrate different speeds (slow, intermediate or

fast). Ex A2 (slowest) < C < S < F < A (fastest)

** since each of the different hemoglobins has

distinctly different amino acid contents, the

different hemoglobins migrate along the acetate

strip at different rates of speed for a specific pH

** the speed at which the hemoglobin travels is

directly dependent on the net harge (because of

the amino acid content)

** the differences in the speed, you can separate

one hemoglobin kind from the other.

3. Upon separation, Hbs is stained and quantitated4. Unknown identified by comparison with known

Hb

** by placing the acetate strip in a densitometer,

one is able to quantify the different hemoglobins

present

** cellulose acetate method is for screening

** citrate agar method is for confirmation; has

an acidic medium

Indications:

- To detect and identify abnormal hemoglobin

III. BONE MARROW EXAMINATION (BONE MARROWASPIRATE AND BIOPSY)

- Marrow fills the spaces between the trabeculaeof bone in the marrow cavity and is soft and

semi-fluid. It is therefore, amendable to

sampling (for smear preparation)

TWO PARTS:

1. ASPIRATE (SMEAR): FOR MORPHOLOGY Cytologic types, proportions of the

hematopoietic cells in the marrow.

2. CORE BIOPSY (HEMOLYTIC REACTIONS) : FORCELLULARITY

Anatomic relation of cells to fat, connectivetissue stroma

Important for evaluating disease thatproduces focal lesions (ex. NHL, MM,

metastatic tumors, amyloidosis, granulomas)

Mandatory for dry taps on aspiration (inaplastic anemia)

This is done under local anesthesia Biopsy usually follows aspiration. This is

performed by changing the direction of the

needle to avoid the aspiration site

COMMON SITES : Adults

1. Posterior superior iliac crest (most common site)2. Anterior superior iliac crest3. Sternum

COMMON SITES: Newborn / infants

1. Upper end of tibial boneSYSTEMATIC APPROACH IN EXAMINING MARROW

SMEAR OR BIOPSY INCLUDE:

1. Cellularity** just examine the proportion of cells to fat

**as one ages the proportion of fat increases as

compared to the number of cells.

2. Myeloid : Erythroid ratio** normal ratios is 3 myeloid elements for every

erythroid elements (3:1)

3. Maturation of erythroid series4. Maturation of myeloid (granulocytic) series5. Number of megakaryocytes (precursor of

platelets)

** normally you should see 1-3 per HPF

6. Other cells: histiocytes, osteoclast, fibroblastand metastatic cells

7. Other abnormalities (granuloma, fibrosis,necrosis and abscess)

** Special stains

o IRON STAIN : iron deficiency anemiao RETICULIN STAIN : assess marrow fibrosis in the

case of myelofibrosis

Indications:

1. Diagnosis or confirmation of certain anemias notpossible by other procuedures

Ex. Iron deficiency anemia, megaloblastic anemia

2. To determine the cause of pancytopeniaEx. Myelodysplastic syndrome, aplastic anemia,

aleukemia leukemia (low wbc count and no

blasts), hypersplenism (problem is no longer in

the bone marrow instead it is the periphery)

3. Diagnosis and classification of leukemias andlymphoid neoplasm

Ex. Acute leukemia, multiple myeloma

4. Staging of lymphoid neoplasm and metastaticcarcinoma


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Ex. Breast cancermetastatic carcinoma in the

bone marrow stage 4

5. Demonstrate focal lesions (granuloma andmetastatic lesions)

SLIDES PICTURES (POWERPOINT) DOUBLE CHECK PLS

NORMOCELLULAR MARROW

- Equal amount or proportion off fat as comparedto marrow on marrow elements

HYPOCELLULAR MARROW

- Increased fat as compared to marrow elements

HYPERCELLULAR MARROW

- May be due to acute leukemia

APLASTIC MARROW (APLASTIC ANEMIA)

- No marrow elements

IDIOPATHIC THROMBOCYTOPENIA PURPURA

- Megakaryocytes are present and it is increasedin ITP

BONE MARROW METASTASIS (MYELOPHTHISIC

ANEMIA)

- Pale staining area with epithelial cells andnormal marrow elements

- @tip of arrow: nests of carcinoid tumor(http://www.va.gov/telepathvisn6/hemcase.htm)

END OF TRANSCRIPTIONAnd He said, My grace is sufficient for you; for My strength is made perfect in

weakness. 2 Corinthians 12:9
http://www.va.gov/telepathvisn6/hemcase.htmhttp://www.va.gov/telepathvisn6/hemcase.htmhttp://www.va.gov/telepathvisn6/hemcase.htmhttp://www.va.gov/telepathvisn6/hemcase.htm

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