Lab Procedures Set 7

Microhematocrit

A measurement of the relative RBC volume for a (small quantity) volume of whole blood spun down in a capillary tube

Microhematocrit Procedure

The capillary tube with whole blood is spun down in a centrifuge, RBCs pack at the bottom, hematocrit is the percent of the tube filled with RBCs.  

What makes up the buffy coat? Is it included in the hematocrit measurement?

WBCs and platelets make up the buffy coat and are not included in the hematocrit measurement.  

Why is the rule of three important?

 

The result has to agree with the rule of three. If it doesn’t, the hemoglobin or hematocrit would be inaccurate.  

Difference between microhematocrit and hematocrit?

*Microhematocrit is considered the manual version of measuring hematocrit vs hematocrit on the analyzer* 

Why is the microhematocrit higher than the automatic hematocrit?

 

Microhematocrit will be a little bit higher than hematocrit because oof the plasma trapped in erythrocytes.  

Erythrocyte Sedimentation Rate

Nonspecific screening test indicating inflammation based on how quickly RBCs stack and settle

Increased ESR associated diseases

Kidney disease, pregnancy, rheumatic fever, rheumatoid arthritis, anemia, syphilis, lupus, thyroid disease, elevated room temperature

Decreased ESR associated diseases

Congestive heart failure, hyper viscosity, decreased fibrinogen, polycythemia, sickle cell anemia 

Westergren method

uses Na citrate diluent with whole blood, read at 60 minutes 

Automated ESR method

on the Alcor, detects rouleaux via flow cell and photometry 

Manual reticulocyte count

finds the bone marrow RBC production index, most effective measure of erythropoiesis, counted out of 1000 RBCs

Decreased Retic associated diseases

Aplastic anemia, exposure to radiation, chronic infection, medications, megaloblastic anemia 

Increased Retic associated diseases

rapid blood loss, high elevation, hemolytic anemia, medications, pregnancy 

Miller eye disc

helps to not count 1000 RBCs, provides standardized area 

How to perform a manual retic

*Mix equal parts stain and whole blood for the manual retic, count all reticulocytes in the large and small square, count small squares until you get to 112 RBCs* 

Manual retic formula

Retic % = Retics in large square / (RBCs in small square \* 9) \* 100 % 

What stain is used for the reticulocyte counts?

 

New Methylene Blue  

Microhematocrit: sources of error

not filling the capillary tube, reading the buffy coat, not following the rule of three 

ESR: sources of error

not keeping the tube free from vibration, not keeping the tube level, not pushing the blood to the zero mark, not replacing the cap 

Manual retic: sources of error

Interferences: nucleated erythrocytes, Howell-jolly bodies, Pappenheimer bodies, cold agglutinins, basophilic stippling, heinz bodies, parasites, autofluorescence 

Percent retic, RPI formula

Retic % = Retics in large square / (RBCs in small square \* 9) \* 100 % 

Conditions associated with increased ESR and retic counts

* Pregnancy  
* Hemolytic anemia  

Conditions associated with decreased ESR and retic counts

Aplastic anemia / sickle cell anemia  

Soucres of error on the hemocytometer

 

Not evenly distributing the object across the hemocytometer.  

\
Overcrowding the hemocytometer.  

Cytospin

concentrates the cells into a smaller area of the slide

Principle of the LAP test

**Helps differentiate CML from a leukemoid reaction**

\
Naphthol AS-BI is hydrolyzed by the enzyme at an alkaline pH. The hydrolyzed substrate with a red-violet LB or fast blue BB dye produces a colored precipitate at the site of enzyme activity

Decreased LAP =

Untreated CML

During a __**Leukemoid reaction**__ LAP changes from…

High normal to increased

High LAP =

Pregnancy, polycythemia vera 

Low LAP =

PNH, Sideroblastic Anemia, Myelodysplastic disorders