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APPROACH TO
ANEMIAS
Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar
Contact: pathologybasics@gmail....
2

OVERVIEW
1. Definition with normal range of Hb and PCV
2. Establishing the presence and severity of anemia
a. determina...
3

*Definition
Reduction in concentration of Hemoglobin below that expected for age and sex matched
healthy controls
Or
Re...
4

*STEP 1: Determination of hemoglobin

(i) Tallquist blotting paper method

Allow blood to absorb into one of the test p...
5

The color of a finger prick blood sample, soaked into chromatography paper, is
compared with the color of known hemoglo...
6

(iv) Cyan meth hemoglobin method (Drabkin’s method)
Recommended by ICSH

Method:
5 ml Drabkin’s reagent + 0.5 ml antico...
7

(v) Oxy Hb method
Ammonium hydroxide (0.04ml / dl) is used to hemolyse the red cells and convert the
hemoglobin to oxyh...
8

(ix) Chemical method
It is an indirect method based on assumption that 1 gm Hb contains approximately 3.47
mg of iron. ...
9

*STEP 2 : Grading of anemia according to Hb levels

Hb levels
(gm/dL)

Normal
13-17 Males
12-15 Females

*Determination...
10

Microhematocrit method:
1. Capillary tubes with coated heparin (75 mm long, 1 mm internal bore) are filled about
3/4th...
11

Normals:

RBC column (PCV)(%)
WBC column (buffy coat) (%)
Plasma column (%)

Normal
40-53 males
36-48 females
0.5-1
50...
12

*STEP 3 Determining the cause of anemia

Peripheral Blood smear

Reticulocyte count

Red cell indices

Serum iron stud...
13

(ii) RED CELL INDICES (MCV, MCH, MCHC AND RDW)
MEAN CORPUSCULAR VOLUME (MCV)
1. Volume of a single RBC
2. Only MCV is ...
14

MEAN CORPUSCULAR HEMOGLOBIN (MCH)
1. It is the average hemoglobin in each RBC
MCH =

Hb (gm/dL)

x

10

RBC count (mil...
15

MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (MCHC)
Definition:
Average concentration of Hemoglobin in a given volume of ...
16

RED CELL DISTRIBUTION WIDTH (RDW)
1. Indicates degree of variation in red cell size
2. Apart from anemias causing vari...
17

(iii) Reticulocyte count
1. Reticulocytes are young RBCs that contain RNA remnants
2. They are stained by supravital s...
18

ARC = Reticulocyte percentage x RBC count in millions/µl
$$ RETICULOCYTE PRODUCTION INDEX (RPI)
1. After formation nor...
19

(iv) Serum iron studies
See notes on iron deficiency anemia

Notes on Laboratory approach to anemias.. By Dr. Ashish V...
20

*STEP 4 MORPHOLOGICAL TYPES flow charts

(i) Macrocytic anemias
MCV >100 fl

Reticulocyte count

Normal (0.5-2.5%)

In...
21

(ii) Microcytic hypochromic anemia
MCV < 80

Serum ferritin

Low
(<12 µg/L)

Normal
(15-300 µg/L)

Iron deficiency ane...
22

(iii) Normocytic normochromic anemia
MCV 80-100 fl

Retic count

High

Normal/Low

Post hemorrhagic
Post hemolytic

Bo...
23

(iv) Hemolytic anemias
Features suggestive of hemolytic anemias
(increased retic, increased indirect bilirubin, low he...
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Laboratory approach to anemias

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this is a series of notes on hematology, useful for undergraduate and post graduate pathology students. Notes have been prepared from standard textbooks and are in a format easy to reproduce in exams.

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Transcript of "Laboratory approach to anemias"

  1. 1. 1 APPROACH TO ANEMIAS Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  2. 2. 2 OVERVIEW 1. Definition with normal range of Hb and PCV 2. Establishing the presence and severity of anemia a. determination of hemoglobin (STEP 1) b. Methods of estimation 1. colorimetric methods i. Visual - Tallquist blotting paper - Sahli’s acid hematin - WHO Hb color scale ii. Using colorimeter - Cyanmeth Hb method - Oxy Hb method - Alkaline method - Haldane method 2. Gasometric methods Van slyke method 3. Chemical methods 4. Specific gravity method c. Grading of anemia (STEP 2) d. determination of hematocrit (PCV) i. Wintrobe method ii. Microhematocrit method 3. Determining the cause of anemia (STEP 3) a. Peripheral blood smear b. Reticulocyte count c. Red cell indices (MCV,MCH,MCHC,RDW) d. Serum iron studies 4. Morphological types flow charts (STEP 4) a. Macrocytic anemia b. Microcytic hypochromic anemia c. Normocytic anemias d. Hemolytic anemias Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  3. 3. 3 *Definition Reduction in concentration of Hemoglobin below that expected for age and sex matched healthy controls Or Reduction in oxygen carrying capacity of blood Normals#: Adult males Adult females Hb 13 – 17 gm/dL 12 – 15 gm/dL PCV 40-50 % 38-45 % #Values vary with age, sex, geographical area and from textbook to textbook. Advisable to determine and set reference values for own lab according to local conditions Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  4. 4. 4 *STEP 1: Determination of hemoglobin (i) Tallquist blotting paper method Allow blood to absorb into one of the test papers and compare with the color scale to determine the percent and weight of hemoglobin in blood under normal and anemic conditions (ii) WHO Hb color scale Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  5. 5. 5 The color of a finger prick blood sample, soaked into chromatography paper, is compared with the color of known hemoglobin content depicted on the scale in 2 g/dl increments from 4 g/dl to 14 g/dl. (iii) Sahli’s acid hematin method Method: 1. The diluent is N/10 Hydrochloric acid (HCL). Add it from the dropping bottle provided to the graduated tube, up to mark 2. 2. Measure 0.2 ml of well-mixed blood, with the provided micropipette (Sahli’s pipette) and transfer it to the HCL in the tube. 3. Thoroughly mix blood and acid using a fine glass rod (HCL will react with the haemoglobin and convert it into acid-haematin, which has a brown color). 4. Wait up to 3 minutes to allow the color to develop sufficiently to achieve an accurate comparison. 5. Add distilled water gradually to the mixture and mix the solution with glass rode. 6. Place the tube in the haemoglobinometer and compare it with the standard. 7. Continue to add distilled water until the sample firstly appears to be detectably pallor than the standard. 8. Note the level of the liquid in the tube. Disadvantages 1. It is tedious and time consuming to perform, especially with large number of samples. It is not accurate (its accuracy is of the order 15 %). Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  6. 6. 6 (iv) Cyan meth hemoglobin method (Drabkin’s method) Recommended by ICSH Method: 5 ml Drabkin’s reagent + 0.5 ml anticoagulated blood 5 min (potassium cyanide+potassium Ferricyanide) Check absorbance Principle: Hemoglobin meth hemoglobin K ferricyanide cyanmethhemoglobin K cyanide Cyanmethhemoglobin is a colored compound. After reaction, absorbance is measured at 540 nm. Absorbance is converted to Hb levels using calculating tables. All forms of hemoglobin (oxy Hb, Carboxy Hb, Meth Hb) except sulphmeth hemoglobin are measured by this method. Drabkin’s reagent is a colored compound, so zero is set using distilled water. Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  7. 7. 7 (v) Oxy Hb method Ammonium hydroxide (0.04ml / dl) is used to hemolyse the red cells and convert the hemoglobin to oxyhemoglobin for measurement in the spectrophotometer. This conversion is complete and immediate and the resulting colour is stable. (vi) Alkaline hematin method Ammonium hydroxide (0.04ml / dl) is used to hemolyse the red cells and convert the hemoglobin to oxyhemoglobin for measurement in the spectrophotometer. This conversion is complete and immediate and the resulting colour is stable. (vii) Haldane method In this method, hemolysis of red cells is produced by mixing blood with a hypotonic solution like distilled water. Carbon monoxide is added to the mixture. The colour of the solution is compared with the standard one. (viii) Gasometric method / Van Slyke method / Manometric method Gasometric method of estimation of hemoglobin by using van Slyke apparatus is the most accurate method. But it is not used routinely in clinical laboratories because it is timeconsuming and the process of estimation is complex. It is used as a reference method to obtain the hemoglobin concentration in blood samples used for standardization of hemoglobin estimation procedures. This is the preferred method for research. If interested in details, please refer to the following article available free online: http://www.jbc.org/content/91/1/307.full.pdf Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  8. 8. 8 (ix) Chemical method It is an indirect method based on assumption that 1 gm Hb contains approximately 3.47 mg of iron. Value of Hb is calculated indirectly from value of iron. (x) Specific gravity method This is the most commonly used method in blood banks for screening blood donors. Principle: The method is based on the fact that plasma or whole blood dropped into a solution of copper sulphate of known gravity is encased in a sack of copper proteinate and the gravity of this discrete drop is not changed for about 15 sec. The rise or fall of the drop during this interval (within 15 sec) shows whether it is lighter or heavier than the solution. Method: 1. The copper sulfate solution is placed into a clear, several inch high test tube that is kept at room temperature and covered to prevent evaporation. A new tube is made daily or after 20-30 tests. 2. A small amount blood is produced from the side of an alcohol swabbed finger using a lancet followed by pressure at the stick site. The blood drop is then drawn into a small capillary tube by capillary action. The finger prick site has gauze applied to it to stop any bleeding. 3. A latex dropper bulb is then attached to the capillary tube containing the blood. The dropper bulb is squeezed slightly to expel a blood drop half an inch above the now opened copper sulfate test tube. The blood drop automatically forms a pellet upon contact with the copper sulfate. The used capillary tube is disposed of as biohazardous waste. Result: The blood drop is observed for a short time (15 sec) to determine whether it sinks (donor hemoglobin above 12.5 g/dL cut-off) or swims (donor hemoglobin MAY be below 12.5 g/dL cut-off). Since the test is just an estimate, many false-negatives tests (hemoglobin is not <12.5 g/dL) are produced and hemoglobin may be checked in another more accurate manner if available. Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  9. 9. 9 *STEP 2 : Grading of anemia according to Hb levels Hb levels (gm/dL) Normal 13-17 Males 12-15 Females *Determination of Hematocrit Mild >10 Moderate 7-10 Severe <7 (PCV) Definition: 1. It is the percentage of blood volume that is occupied by red cells. 2. It is expressed as a percentage. Uses: 1. It is used to detect anemia and polycythemia 2. To calculate red cell indices such as MCV or MCHC 3. To check accuracy of Hb value Methods: 1. Wintrobe’s method 2. Microhematocrit method Wintrobe’s method: 1. Anticoagulated blood is centrifuged for 30 min at 2300 g in a wintrobe’s tube 2. Blood gets separated as shown below 3. Wintrobe tube is 110 mm long and has an internal bore of 3mm diameter and is closed at one end Wintrobe tube rack after centrifugation Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  10. 10. 10 Microhematocrit method: 1. Capillary tubes with coated heparin (75 mm long, 1 mm internal bore) are filled about 3/4th with blood, sealed at one end with bees wax. Bee’s wax plate 2. They are centrifuged in a capillary centrifuge for 5 min 3. Readings are obtained either via a microhematocrit rube reading device or Arithmetic graph paper Microhematocrit tube Arithmetic graph paper Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  11. 11. 11 Normals: RBC column (PCV)(%) WBC column (buffy coat) (%) Plasma column (%) Normal 40-53 males 36-48 females 0.5-1 50-55, straw colored Abnormals: RBC column (PCV)(%) WBC column (buffy coat) (%) Plasma column (%) Value Anemia <40 males <36 females Polycythemia >53 males >48 females >1 – leukocytosis, thrombocytosis, leukemia This layer can be pipetted and used to demonstrate malarial parasites and blast cells Pink – hemolysis Yellow – jaundice Colorless - anemia RULE OF THREE RBC COUNT (NORMAL 5) X 3 = HEMOGLOBIN (NORMAL 15) HEMOGLOBIN (15) X 3 = PCV (NORMAL 45) Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  12. 12. 12 *STEP 3 Determining the cause of anemia Peripheral Blood smear Reticulocyte count Red cell indices Serum iron studies (i) PERIPHERAL BLOOD SMEAR Only salient points related to RBCs will be discussed here. For details please refer to separate notes on Peripheral blood smear examination. Microcytic, hypochromic Sickle cells Oval macrocytes Spherocytes Target cells Schistocytes Burr cells Bite cells Tear drop cells Polychromatic RBC Basophilic stippling Howel jolly bodies Rouleaux formation nRBC Autoagglutination Iron deficiency anemia, thalessemia Sickle cell anemia Megaloblastic anemia, alcoholism Heriditary spherocytes, autoimmune hemolysis Thalessemia, jaundice, HbC disease Microangiopathic hemolytic anemia Uremia G6 PD deficiency Myelofibrosis, myelopthisic anemia Hemolysis, blood loss Lead poisoning (coarse) megaloblastic anemia (fine) Megaloblastic anemia, thalessemia, post splenectomy Multiple myeloma, hypergammaglobulinemia Hemolytic anemia AIHA (cold antibody type) Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  13. 13. 13 (ii) RED CELL INDICES (MCV, MCH, MCHC AND RDW) MEAN CORPUSCULAR VOLUME (MCV) 1. Volume of a single RBC 2. Only MCV is determined by a cell counter, other parameters are calculated from PCV and MCV MCV = PCV X RBC COUNT 10 Normals: MCV 80-100 fL Classification of anemias based on MCV Microcytic anemia <80 fl 1. Iron deficiency anemia 2. Thalessemia 3. Sideroblastic anemia 4. Anemia of chronic diseases Normocytic anemia 80-100 fl Decreased retic count 1. Aplastic anemia 2. anemia of chronic diseases 3. Chronic renal failure 4. hypothyroidism 5. myelopthisic anemia Increased retic count 1. Acute blood loss 2. Hemolytic anemia Macrocytic anemia >100 fl Megaloblastic anemia 1. Vit B12 deficiency 2. Folate deficiency Non megaloblastic 1. Liver disease 2. Alcoholism 3. MDS 4. hypothyroidism Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  14. 14. 14 MEAN CORPUSCULAR HEMOGLOBIN (MCH) 1. It is the average hemoglobin in each RBC MCH = Hb (gm/dL) x 10 RBC count (millions/µl) Normals: MCH 27-32 pg Abnormals: Low MCH Microcytic hypochromic anemia High MCH Macrocytic anemia Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  15. 15. 15 MEAN CORPUSCULAR HEMOGLOBIN CONCENTRATION (MCHC) Definition: Average concentration of Hemoglobin in a given volume of packed red cells MCHC = Hb (gm/dL) x 100 PCV Normals: MCHC 32-36 gm/dL Abnormals: Low MCHC Microcytic hypochromic anemia High MCHC 1. hereditary spherocytosis 2. >40 gm% - cold agglutinin disease, MPD, viral infection Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  16. 16. 16 RED CELL DISTRIBUTION WIDTH (RDW) 1. Indicates degree of variation in red cell size 2. Apart from anemias causing variation in cell size, also useful to differentiate iron deficiency anemia from thalessemia minor (RDW raised, MCV low – iron def) (RDW normal, MCV low – thalessemia minor) Normals: RDW 11.6-14.6 % Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  17. 17. 17 (iii) Reticulocyte count 1. Reticulocytes are young RBCs that contain RNA remnants 2. They are stained by supravital stains like brilliant cresyl blue or new methylene blue 3. used to find out the erythropoietic activity of bone marrow and to differentiate aplastic anemias from other types of anemias $$ RETIC COUNT Retic count = Reticulocytes counted x 100 No. of RBCs counted Normals: Adults New born 0.5 - 2.5% 2 - 5% Abnormals: Reticulocytosis 1. acute blood loss 2. hemolytic anemia 3. response to therapy in nutritional anemias Reticulocytopenia Decreased production: 1. Iron deficiency anemia 2. Anemia of chronic disease 3. Aplastic anemia 4. Anemia due to marrow infiltration (leukemia, lymphoma, mets) Ineffective erythropoeisis: $$ CORRECTED RETICULOCYTE COUNT 1. Reticulocyte count depends on PCV of the patient, low PCV can give falsely low retic count and vice versa. 2. Hence retic count is corrected for normal average PCV for age of the patient Corrected Retic count = Retic count x PCV Avg PCV for age $$ ABSOLUTE RETICULOCYTE COUNT Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  18. 18. 18 ARC = Reticulocyte percentage x RBC count in millions/µl $$ RETICULOCYTE PRODUCTION INDEX (RPI) 1. After formation normally reticulocytes spend 2 days in bone marrow and one day in peripheral blood before fully maturing 2. When there is over production , they are released prematurely and they require more time for maturation in peripheral blood. This results in doubling of reticulocytes in blood 3. So RPI is calculated to get an idea about the actual erythropoeitic activity of bone marrow. Reticulocyte production index = corrected reticulocyte count Maturation time in days MATURATION TIME DEPENDS ON PCV PCV >35 25-35 15-25 5-15 TIME (DAYS) 1 1.5 2 2.5 Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  19. 19. 19 (iv) Serum iron studies See notes on iron deficiency anemia Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  20. 20. 20 *STEP 4 MORPHOLOGICAL TYPES flow charts (i) Macrocytic anemias MCV >100 fl Reticulocyte count Normal (0.5-2.5%) Increased Bone marrow Reticulocytosis in hemolytic anemia Megaloblastic Normoblastic dysplastic Hypocellular Folate/B12 def Liver disease Hypothyroidism MDS Aplastic anemia Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  21. 21. 21 (ii) Microcytic hypochromic anemia MCV < 80 Serum ferritin Low (<12 µg/L) Normal (15-300 µg/L) Iron deficiency anemia High (>300 µg/L) Electrophoresis Sideroblastic Anemia Increased HbA2/HbF Normal Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  22. 22. 22 (iii) Normocytic normochromic anemia MCV 80-100 fl Retic count High Normal/Low Post hemorrhagic Post hemolytic Bone marrow Normal Abnormal Anemia of chronic Diseases Chronic renal Failure aplastic anemia Myelopthisic anemia Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes
  23. 23. 23 (iv) Hemolytic anemias Features suggestive of hemolytic anemias (increased retic, increased indirect bilirubin, low hemoglobin) Examine peripheral smear Malaria Normal Cells G6pd PNH Unstable Hb Sickle cells Bite cells G6PD Schistocytes Spherocytes MAHA Microcytic Hypochromic DAT Thalessemia Positive AIHA DIC Negative Spherocytosis HUS TTP RENAL NEUROLOGIC HEMOGLOBIN ELECTROPHORESIS Notes on Laboratory approach to anemias.. By Dr. Ashish V. Jawarkar Contact: pathologybasics@gmail.com website: pathologybasics.wix.com/notes

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